Nucleic acid separation using immobilized metal affinity chromatography

ABSTRACT

An immobilized metal affinity chromatography (IMAC) method for separating and/or purifying compounds containing a non-shielded purine or pyrimidine moiety or group such as nucleic acid, presumably through interaction with the abundant aromatic nitrogen atoms in the purine or pyrimidine moiety. The method can also be used to purify compounds containing purine or pyrimidine moieties where the purine and pyrimidine moieties are shielded from interaction with the column matrix from compounds containing a non-shielded purine or pyrimidine moiety or group. Thus, double-stranded plasmid and genomic DNA, which has no low binding affinity can be easily separated from RNA and/or oligonucleotides which bind strongly to metal-charged chelating matrices. IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a ribozyme, and remove primers and other contaminants from PCR reactions. The metal ion affinity of yeast RNA decreases in the order: copper (II), nickel (II), zinc (II), and cobalt (II).

RELATED APPLICATIONS

This application is a Divisional of and claims priority of U.S. patentapplication Ser. No. 09/994,701 filed Nov. 6, 2001 now U.S. Pat. No.7,598,371, which claims priority of U.S. Provisional Patent ApplicationSer. No. 60/246,292 filed Nov. 6, 2000.

RELATED APPLICATIONS

This application claims provisional priority to U.S. Provisional PatentApplication Ser. No. 60/246,292 filed Nov. 6, 2000.

GOVERNMENT LICENSE RIGHTS STATEMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grant No.R825354-01-0 from the National Space Biomedical Research Institute andthe Environmental Protection Agency.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an immobilized metal affinitychromatography (IMAC) instrument, a substrate containing immobilizedmetal affinity ligands, and a method for purifying and/or separatingcompounds containing a non-shielded purine or pyrimidine moiety or groupusing the instrument or substrate.

More particularly, the present invention relates to an immobilized metalaffinity chromatography (IMAC) instrument and/or a substrate containingimmobilized metal affinity ligands where the metal ion immobilized onthe ligand of the column of an IMAC instrument or the substrate iscapable of binding compounds containing a non-shielded purine orpyrimidine moiety or group where binding affinities results in aseparation of different compounds containing a non-shielded purine orpyrimidine moiety or group or in a purification of compounds that do notcontain a non-shielded purine or pyrimidine moiety or group fromcompounds that do contain a non-shielded purine or pyrimidine moiety orgroup. The present invention also relates to a method for purifyingand/or separating compounds containing a non-shielded purine orpyrimidine moiety or group such as single stranded DNA, RNA, or othercompounds containing a non-shielded purine or pyrimidine moiety orgroup, or to a method for removing compounds containing a non-shieldedpurine or pyrimidine moiety or group from compounds that do not containa non-shielded purine or pyrimidine moiety or group such as removingnucleotides and primers from PCR reactions and/or reaction products andpurifying plasmid DNA.

2. Description of the Related Art

Immobilized Metal Affinity Chromatography (IMAC) was introduced byPorath et al. (1, 2) as a means of purifying proteins based on theaffinity of their surface-exposed amino acids (especially histidines)for chelated metal ions. The method has found widespread application inthe purification of recombinant histidine-tagged and pharmaceuticalproteins, most commonly using Cu(II) and Ni(II) ions chelated byiminodiacetic acid (IDA) and nitrilotriacetic acid (NTA)functionalities. Metal chelate ligands are best known as affinity agentsin chromatography but have also been immobilized on foams (3), membranes(4), biosensor chips (5), and in electrophoresis gels (6); they havealso been used as affinity precipitation agents (7).

The interaction of metal ions with nucleic acids is a long-standing andactive field of study (8, 9). While metal ion binding to nucleic acidsis well known, and plays an important role in the function of the widelyused cancer drug cisplatin (10), IMAC has found very limited applicationin the purification of nucleic acids. Fanou-Ayi and Vijayalakshmi (11)and Hubert and Porath (12) demonstrated the binding of mononucleotidesto copper IMAC resins, and histidine-conjugated PCR primers have beenused to facilitate purification of the resulting histidine-taggedoligonucleotide products (13), but the potential applications of IMAC innucleic acid separation and analysis remain largely unexplored.

Thus, there is a need in the art for an IMAC instrument and method,where the IMAC column includes metals or metal ions capable of bindingcompounds containing a non-shielded purine or pyrimidine moiety or groupto effectuate separation and/or purification of different compoundscontaining a non-shielded purine or pyrimidine moiety or group or forpurifying compounds that do not contain a non-shielded purine orpyrimidine moiety or group from compounds that do contain a non-shieldedpurine or pyrimidine moiety or group.

SUMMARY OF THE INVENTION

The present invention provides a composition including a first molecularcomponent having immobilized metal atoms and/or ions and a secondmolecular component including a non-shielded purine moiety or groupand/or pyrimidine moiety or group, where the second molecular componentis bound to, associated with and/or interacting with the metal atomsand/or ions of first molecular component.

The present invention provides a composition including a molecularcomponent having immobilized metal atoms and/or ions capable ofassociating with, interacting with or binding a second molecular comSEQ. ID NO. 1 5′ TAATTGTTGCCGGGAAGCTAGAG 3′ and SEQ. ID NO. 2 5′TCGCATTGAATTATGTGCTGTGTAG 3′(MWG Biotech) component including anon-shielded purine moiety or group and/or pyrimidine moiety or group.

The present invention provides an immobilized metal affinity compositionincluding a molecular component including immobilized metal atoms and/orions capable of binding compounds including a non-shielded purine moietyor group and/or pyrimidine moiety or group.

The present invention provides an immobilized metal affinity compositionincluding a molecular component including immobilized metal atoms and/orions capable of binding compounds including a non-shielded purine moietyor group and/or pyrimidine moiety or group and a compound including anon-shielded purine moiety or group and/or pyrimidine moiety or groupbound thereto.

The present invention provides an immobilized metal affinitychromatography (IMAC) column including a matrix or absorbent havingimmobilized metal atoms and/or ions capable of binding compoundscontaining a non-shielded purine moiety or group and/or pyrimidinemoiety or group.

The present invention provides an immobilized metal affinitychromatography (IMAC) column including a matrix or absorbent havingimmobilized metal atoms and/or ions capable of binding compoundscontaining a non-shielded purine moiety or group and/or pyrimidinemoiety and/or group and a compound containing a non-shielded purinemoiety or group and/or pyrimidine moiety or group bound thereto.

The present invention provides a surface including an immobilized metalaffinity composition coated thereon, where the composition includesimmobilized metal atoms and/or ions capable of binding compoundscontaining a non-shielded purine moiety or group and/or pyrimidinemoiety or group.

The present invention provides a surface including an immobilized metalaffinity composition coated thereon, where the composition includesimmobilized metal atoms and/or ions capable of binding compoundscontaining a non-shielded purine moiety or group and/or pyrimidinemoiety or group, and a compound containing a non-shielded purine moietyor group and/or pyrimidine moiety or group bound thereto.

The present invention provides a method for separating differentcompounds containing a non-shielded purine moiety or group and/or apyrimidine moiety or group by passing a solution containing thedifferent compounds through an immobilized metal affinity chromatography(IMAC) column including immobilized metal atoms and/or ions capable ofbinding compounds containing a non-shielded purine moiety or groupand/or pyrimidine moiety or group and analyzing the effluent from thecolumn as a function of time or over a given period of time.

The present invention provides a method for purifying a solutioncomprising a major compound having one or more (at least one) shieldedpurine moiety or group or pyrimidine moiety or group and a minorcompound containing one or more (at least one) non-shielded purinemoiety or group or pyrimidine moiety or group by passing the solutionthrough an immobilized metal affinity chromatography (IMAC) columnincluding a matrix having immobilized metal atoms and/or ions capable ofbinding compounds containing one or more (at least one) non-shieldedpurine moiety or group or pyrimidine moiety or group and analyzing theeffluent from the column as a function of time or over a given period oftime. The column can include metal atoms and/or ions that have a higheror lower binding affinity for the major verses the minor compound andseparation will result, provided that the binding affinities are indeeddifferent.

The present invention provides a method for purifying a solutioncontaining a compound that does not contain a non-shielded purine moietyor group or pyrimidine moiety or group and a compound that does containa (or a plurality of) non-shielded purine moiety(ies) or group(s) and/ora (or a plurality of) pyrimidine moiety(ies) or group(s) by passing thesolution through an immobilized metal affinity chromatography (IMAC)column including immobilized metal atoms or ions capable of bindingcompounds containing a non-shielded purine moiety or group and/orpyrimidine moiety or group and analyzing the effluent from the column asa function of time or over a given period of time.

The present invention also provides a method including the steps ofcontacting a mixture including components having shielded one or morepurine and/or pyrimidine moieties and components having non-shielded oneor more purine and/or pyrimidine moieties with a molecular componentincluding immobilized metal atoms and/or ions capable of binding thecomponents having one or more non-shielded purine and/or pyrimidinemoieties and eluting the components having one or more shielded purineand/or pyrimidine moieties.

The present invention also provides a method including the steps ofcontacting a mixture of components having non-shielded one or morepurine and/or pyrimidine moieties with a molecular component includingimmobilized metal atoms and/or ions capable of binding the componentshaving one or more non-shielded purine and/or pyrimidine moieties toform an associated mixture and flowing a solution over the associatedmixture to affect a partial or complete separation of components.

The present invention also provides a composition comprising a polymericmaterial including immobilized metal atoms and/or ions and a compoundhaving a non-shielded purine and/or pyrimidine moiety or group boundthereto, where the compound is selected from the group of mRNA, RNA,ribosomyl DNA, denatured DNA, denatured cDNA, and other biologicalmolecules having a purine and/or pyrimidine moiety or group.

The present invention also provides an immobilized metal affinitycomposition comprising immobilized metal atoms or ions capable ofbinding compounds containing a non-shielded purine or pyrimidine moietyor group and a compound containing a non-shielded purine or pyrimidinemoiety or group bound thereto, where the compound is selected from thegroup of mRNA, RNA, ribosomyl DNA, denatured DNA, denature cDNA, andother biological molecules having a purine and/or pyrimidine moiety orgroup.

The present invention also provides an immobilized metal affinitychromatography (IMAC) column comprising immobilized metal atoms or ionscapable of binding compounds containing a non-shielded purine orpyrimidine moiety or group and a compound containing a non-shieldedpurine or pyrimidine moiety or group bound thereto, where the compoundis selected from the group of mRNA, RNA, ribosomyl DNA, denatured DNA,denature cDNA, and other biological molecules having a purine and/orpyrimidine moiety or group.

The present invention also provides a surface comprising an immobilizedmetal affinity composition coated thereon, where the compositionincludes immobilized metal atoms or ions capable of binding compoundscontaining a non-shielded purine or pyrimidine moiety or group, and acompound containing a non-shielded purine or pyrimidine moiety or groupbound thereto, where the compound is selected from the group of mRNA,RNA, ribosomyl DNA, denatured DNA, denature cDNA, and other biologicalmolecules having a purine and/or pyrimidine moiety or group.

The present invention also provides a multisubstrate column comprising afirst zone comprising an IMAC ligand or matrix and a second zonecomprising another separation medium, where the IMAC, which isrelatively insensitive to an ionic strength of a solution, bindscompounds comprising non-shielded purine or pyrimidine moieties orgroups and the second zone is designed to separate other constituents ofthe solution based on the other constituents interaction with thestationary material of the second zone. The first zone is followed by oris preceded by the second zone. Preferably, the second zone follows thefirst zone, where the second zone comprises non-metal contain IMACsubstrate for binding any leached metal ions from the first zone.Alternatively, the second zone comprises an anion exchange material orHIC material. Preferably, the column is made using monolith technology.

The present invention also provides an IMAC pre-filters for purifyingsolution containing large quantities of plasmid.

The present invention also provides an IMAC ligand or matrices depositedon capillary walls or in solution, where the IMAC ligand or matrix wouldalter the retention times of compounds having non-shielded purine and/orpyrimidine moieties in capillary electrophoresis. Preferably, thesolution comprises an IMAC ligand or matrix bonded to, associated withor deposited on substrate. Preferably, the substrate is a polymer ordendrimer. Preferably, the polymer is a PEG.

The present invention also provides an IMAC ligand or matrices depositedon a surface or in a gel, where the IMAC ligand or matrix altersretention times for of compounds having non-shielded purine and/orpyrimidine moieties in slab gel electrophoresis or alters in traditionalIMAC chromatography.

The present invention also provides a plasmid separation technique usingmembranes coated with or impregnated with an IMAC matrix to filter outor prevent free RNA from migrating to the other side of the membranestructure.

The present invention also provides a porous stirrer (a stick-like rod)with an IMAC ligand deposited thereon or therein to batch bind RNA froma solution containing plasmids to clarify plasmid lysate.

The present invention also provides an assay using IMAC matricesdeposited in the wells of microplates and then bind a single strandedoligonucleotide having a fluorescent tag to the matrix, where the IMACmatrix either contains a quencher in close proximity to the metalbinding sites or the substrate upon which the IMAC is coated acts as afluorescent quencher and when a complimentary sequence is added to awell containing the fluorescent tagged oligonucleotide, the two pair andthe paired sequence is released and the tag fluoresces.

The present invention also provides an assay comprising the steps ofcontacting a microplate substrate comprising wells coated with acomposition comprising an IMAC ligand with a single strandedoligonucleotide including a first molecular and/or atom tag to form aIMAC-oligonucleotide complex, contacting a nucleic acid sequenceincluding a second molecular and/or atomic tag with theIMAC-oligonucleotide complex, and measuring a fluorescence of theresulting mixtures, where the tags interact to produce a fluorescentpair or a non-fluorescent pair if the nucleic acid sequence includes acomplimentary subsequence to oligonucleotide and where pair results inrelease of the paired nucleic acid sequence and oligonucleotide.

The present invention also provides an assay comprising the steps ofcontacting a substrate comprising a surface coated with a compositioncomprising an IMAC ligand and a first fluorophore with anoligonucleotide including a second fluorophore and measuring aneffective Stoke shift such that a large effective Stoke shift signifiesoligonucleotide binding to the coated substrate and a normal effectiveStoke shift signifies no oligonucleotide binding to the coatedsubstrate.

The present invention also provides a method for separating differentcompounds containing a non-shielded purine or pyrimidine moiety or groupby passing a solution containing the different compounds through animmobilized metal affinity chromatography (IMAC) comprising immobilizedmetal atoms or ions capable of binding compounds containing anon-shielded purine or pyrimidine moiety or group and analyzing theeffluent from the column as a function of time or over a given period oftime.

The present invention also provides a method for purifying a solutioncontaining a major compound containing a non-shielded purine orpyrimidine moiety or group and a minor compound containing anon-shielded purine or pyrimidine moiety or group by passing thesolution through an immobilized metal affinity chromatography (IMAC)comprising immobilized metal atoms or ions capable of binding compoundscontaining a non-shielded purine or pyrimidine moiety or group andanalyzing the effluent from the column as a function of time or over agiven period of time. The column can include metal atoms or ions thathave a higher or lower binding affinity for the major verses the minorcompound and separation will results, provided that the bindingaffinities are indeed different.

The present invention also provides a method for purifying a solutioncontaining a compound that does not contain a non-shielded purine orpyrimidine moiety or group and a compound that does contain anon-shielded purine or pyrimidine moiety or group by passing thesolution through an immobilized metal affinity chromatography (IMAC)comprising immobilized metal atoms or ions capable of binding compoundscontaining a non-shielded purine or pyrimidine moiety or group andanalyzing the effluent from the column as a function of time or over agiven period of time.

The present invention also provides a method for removing fluorophoretagged nucleotides or nucleosides from a solution involving passing thesolution through an IMAC column capable of either binding to nitrogenatoms of a base residue in the nucleotides or nucleoside, binding toatoms and/or functional groups of the fluorophores or both and removingall non-bound materials from the column resulting in the removal offluorophore labeled nucleotides or oligonucleotides from the solution.

The present invention also provides a method for making multisubstratecolumns comprising running a small amount of IMAC ligand onto anactivated column and then flooding the rest of the column with one ormore additional ligands or stationary phases.

The present invention also provides a method for separating compoundscomprising the steps of: (a) passing a solution comprising a mixture ofcompounds including a non-shielded purine or pyrimidine moiety or groupthrough a column including an interior surface coated with animmobilized metal affinity composition comprising a matrix andimmobilized metal ions capable of binding compounds containing anon-shielded purine or pyrimidine moiety or group; (b) analyzing theeffluent from the column as a function of time for each compound; and(c) collecting more purified samples of each compound.

The present invention also provides a method for separating compoundscomprising the steps of (a) passing a solution comprising a mixture ofcompounds including a non-shielded purine or pyrimidine moiety or groupthrough a column including an interior surface coated with animmobilized metal affinity composition comprising a matrix andimmobilized metal ions capable of binding compounds containing anon-shielded purine or pyrimidine moiety or group; (b) detecting thepresent of each compound in an effluent from the column as a function oftime from detectable properties of each compound; and (c) determiningthe identity of each compound from the detected properties.

The present invention also provides a method for separating compoundscomprising the steps of: (a) contacting a solution comprising a firstcompound including a non-shielded purine or pyrimidine moiety or groupand a second compound including no non-shielded purine or pyrimidinemoieties or groups with an immobilized metal affinity compositioncomprising a matrix and immobilized metal ions capable of bindingcompounds containing a non-shielded purine or pyrimidine moiety orgroup; and (b) washing the second compound from the matrix to form amore purified second compound.

The present invention also provides a method for separating compoundscomprising the steps of: (a) passing a solution comprising a firstcompound including a non-shielded purine or pyrimidine moiety or groupand a second compound including no non-shielded purine or pyrimidinemoieties or groups through a column coated with an immobilized metalaffinity composition comprising a matrix and immobilized metal ionscapable of binding compounds containing a non-shielded purine orpyrimidine moiety or group; and (b) collecting the second compound in amore purified state.

The present invention also provides a method for separating compoundscomprising the steps of: (a) passing a solution comprising a firstcompound including a non-shielded purine or pyrimidine moiety or groupand a second compound including no non-shielded purine or pyrimidinemoieties or groups over a surface coated with an immobilized metalaffinity composition comprising a matrix and immobilized metal ionscapable of binding compounds containing a non-shielded purine orpyrimidine moiety or group; and (b) collecting the second compound in amore purified state.

The present invention also provides a method for purifying a solutioncontaining a major compound containing a non-shielded purine orpyrimidine moiety or group and a minor compound containing anon-shielded purine or pyrimidine moiety or group by passing thesolution through an immobilized metal affinity chromatography (IMAC)including immobilized metal atoms or ions capable of binding compoundscontaining a non-shielded purine or pyrimidine moiety or group andanalyzing the effluent from the column as a function of time or over agiven period of time. The column can include metal atoms or ions thathave a higher or lower binding affinity for the major verses the minorcompound and separation will results, provided that the bindingaffinities are indeed different.

The present invention also provides a method for purifying a solutioncontaining a compound that does not contain a non-shielded purine orpyrimidine moiety or group and a compound that does contain anon-shielded purine or pyrimidine moiety or group by passing thesolution through an immobilized metal affinity chromatography (IMAC)including immobilized metal atoms or ions capable of binding compoundscontaining a non-shielded purine or pyrimidine moiety or group andanalyzing the effluent from the column as a function of time or over agiven period of time.

The present invention also provides a method for separating poly(A)tailed mRNA from eukaryotic cells comprising the step of passing asolution containing eukaryotic cell mRNA through an IMAC column or overan IMAC matrix and separating poly(A) tailed mRNA from other mRNA, wherethe poly(A) tailed mRNA elutes from the IMAC matrix last because thepoly(A) tail has such a high affinity for IMAC matrix, where the poly(A)tails are usually 50-200 bases long.

The present invention also provides a method for separating denaturednucleic acid sequences having A rich regions from the complementarystranded having T rich regions comprising contacting a solutioncontaining the sequences having A rich regions with an IMAC matrix orligand and separating all unbound components in the solution andcollecting the sequences having A rich regions.

The present invention also provides a method for separating denaturednucleic acid sequences having C rich regions from the complementarystranded having G rich regions comprising contacting a solutioncontaining the sequences having C rich regions with an IMAC matrix orligand and separating all unbound components in the solution andcollecting the sequences having C rich regions.

The present invention also provides a method for separating denaturednucleic acid sequences having A-C, A-G, and/or A-C-G rich regions fromthe complementary stranded having T-G, T-C and or T-G-C rich regionscomprising contacting a solution containing the sequences having A-C,A-G, and/or A-C-G rich regions with an IMAC matrix or ligand andseparating all unbound components in the solution and collecting thesequences having A-C, A-G, and/or A-C-G rich regions.

The present invention also provides a method for purifying food stuffscontaining purine and/or pyrimidine moieties comprising the steps of (a)forming a crude food stuff comprising cellular constituents includingdigestable proteins and nucleic acid contaminants; (b) contacting thefood stuff with an agent including an IMAC ligand coordinating a metalatom and/or ion capable of binding contaminants including a non-shieldedpurine or pyrimidine moiety to form an IMAC-contaminant composition; and(c) separating the IMAC-contaminant composition from the crude foodstuff to produce a purified food stuff. Further, the method includes thestep of treating the crude food stuff with a DNASE, endo or exo nucleaseor other nucleic acid digestion enzyme or agent prior to the contactingstep.

The present invention also provides a method for purifying a crudecompound containing a non-shielded purine and/or pyrimidine moietycomprising the steps of (a) forming a crude mixture comprising a desiredcompound and acid contaminants; (b) contacting the crude mixture with anagent including an IMAC ligand coordinating a metal atom and/or ioncapable of binding to the desired compound to form an IMAC-compoundcomplex; (c) separating the complex from the contaminants; and (d)recovering the compound from the complex. Preferably, the compound is anAIDS drugs, co-enzyme A, or the like. The preferred AIDs drugs includeAZT or DDI.

Preferably, the interactions between the metal atoms and/or ions and thenon-shielded purine and/or pyrimidine are reversible.

Tabulation of Applications and Preferences

TABLE-US-00001 TABLE A Parameter Units/Preferred More Prefer. Most Pref.Product % 95-100 99-100 99.99 purity RNA in DNA % of ppt. 0-10 0-2 0-1DNA Product Type pDNA, mDNA, cDNA, pDNA, mRNA, pDNA oDNA, genomic DNA,genomic DNA, Primer, Oligonucleotide, oligonucleotides mRNA, PCRproducts A-tailed PCR products, polyA mRNA, RNA-reduced solution,DNA-reduced solution, Nucleotide-reduced solution, Plasmid DNA withreduced content of nicked and linearized forms, Double-stranded hybridsColumn .degree. C. 0-120 5-50 15-25 Temperature for Separation MetalsMetal Cu(II), Ni(II), Zn(II), Co(II), Cu(II), Ni(II) Cu(II) Sc(II),Ti(II), V(II), Cr(II), Mn(II), Fe(II) NonShielded Groups Bases insingle-stranded Bases in single-Bases in single-nucleic acids with1-5,000,000 stranded stranded bases nucleic acids nucleic acids with4-50,000 with 10-5,000 bases bases Purines Compounds Adenine, guanine NNneed Adenine, adenine more non-nucleic acid guanine examplesNNPyrimidines Compounds Cytosine, thymine, uracil Support type Agarose,acrylamide, silica, Agarose, silica agarose Material other polymers,“Smart polymer” Silica, silicon, glass, dextran, polystyrene,phase-separating polymer bearing chelator Column Volume cc/g product 1to 100,000 10-1,000 100 Space Velocity Vol/Vol/Hr. NNN 0-infinite (fornon-flow 1-1,000 5 applications, or zero-volume layers of adsorbent)Support Shape Beads, cut pellets, well plate, Beads, magnetic beadsShape monolith, filter, membrane, particles, well tube, spin column,MEMS plate device, magnetic particles, thermo- or salt-precipitablepolymer, phase-partitioning polymer, soluble chelating polymer Metal.mu.mol/ml 10-100 30-100 50-100 Concentration Matrix Binding Major:minor1.001 to 10000000 3 to 10,000 30 Affinity compound Fluorophores Com'd,Fluorescein, Texas Red, Fluorescein, Fluorescein Mfger Cy3, Cy5,rhodamine, Cy5, Cy3, ALEXA dyes, quantum dot ALEXA dyes Fluorophoresg/liter 1 fM-1 M 1 nM-1 mM 50 uM Conc. Nucleotides Compounds A, G, C, T,I A, G A Nucleosides Compounds A, G, C, T, I A, G A 2nd Zone CompoundsHydrophobic Interaction Other Chromatography, Ion Constituents Exchange,Hydroxyapatite, Fe(II) IMAC, Butyl Agarose, Reverse Phase Resin,Zirconia Other Area Filter, mesh, frit, adsorbent, frit, adsorbent,Adsorbent surfaces for coating on column wall, coating on coating onIMAC Ligand coating on tubing wall, column wall wall coating on tank orcontainer wall HIC/Ion exch. Type Butyl, phenyl, amine, QuaternaryQuaternary Material Quaternary amine, DEAE, amine, DEAE amine C18(reverse phase) Monoliths Shape Rod, chip, disk, hollow tube Disk, roddisk IMAC Ligands Iminodiacetic acid (IDA), IDA, NTA IDA LigandsNitrilotriacetic acid (NTA), Pentadentate chelator (PDC),tris-(2-ethylaminoethyl)amine (TREN), dipicolyl amine (DPA), chelatinglipids Prefilters Type Spin, precoat, RNA removal Matrix CompositionPolymer with chelating groups, NTA agarose, IDA on acrylamide polymer,chelating silane derivatives on silica Detectors Type Flow absorbancedetector, flow fluorescence detector, flow electrochemical detector,Spectrometer, GLC, fluorometer, fluorescence imaging, Mass Spectrometer,plasmon resonance, microbalance, electrode Washing Composition Water,ethanol, polyethylene Fluid glycol, purine solution, imidazole, ammoniumchloride, histidine, AMP, ADP, ATP, GTP, GDP, GMP, Guanine, Adenosine,competitive eluant, base, acid, amine Membranes Type, MfgerSemi-permeable membranes with IMAC Ligands attached.sup.a,b Comp'd w/oStructure Structured plasmid DNA, Plasmid DNA, Plasmid DNA non shieldedgDNA Structured RNA Defect-free Purine/Pyrimidine (some types)Defect-free PCR product PCR product, RNA/DNA hybrid Comp'd WithCompounds Denatured plasmid mRNA, PCR RNA, primer non shielded DNA,Denatured gDNA products with Purine/Pyrimidine mRNA Defect-containingdefects, RNA in PCR product, PCR product alkaline lysate, with extraoverhang, RNA primer in alkaline lysate, RNA/DNA hybrid, sequencingladder, primer, nucleotide, mismatch-containing duplex Poly A Tail Bases1-1000 50-200 100 Length A Rich Region Compounds PolyA, mRNA T RichRegion Compounds PolyT, mRNA G Rich Compounds PolyG, mRNA Region C/URich Compounds PolyU, PolyU, mRNA Region Polyethylene Mers 2000, 6000,8000, 10000, etc. 6000-8000 Glycol (PEG) Dendrimer Types TenticalChromatography media (higher product capacties when IMAC ligand is on adendrimer linker) Gels Types Polyacrylamide, Agarose, Food Stuffs TypesSingle cell protein, removal of nucleic acids from food stuffs ingeneral. Digestion Types DNASE, endo-nuclease, exo-Enzymes nucleaseProducts Compounds AIDS drugs comprising AZT, DDI, coenzyme A, acyclicpurine nucleoside analogs (e.g. Acyclovir). .sup.aChai, S. A., R. R.Beitle, and M. R. Coleman, “Facilitated Transport Metal AffinityMembranes,” International Journal of Biochromatography, 2, 125-131,1996. .sup.bOxford, C. A. Taylor, R. R. Beitle, and M. R. Coleman,“Effect of Chelated Metal on Amino Acid Transport in FaciliatedTransport Membranes Incorporating Metal Affinity,” ACS Symposium onChemistry and Material Science of Synthetic Membranes, 1999.

DESCRIPTION OF THE DRAWINGS

The invention can be better understood with reference to the followingdetailed description together with the appended illustrative drawings inwhich like elements are numbered the same:

FIG. 1 graphs different isotherm binding curves for baker's yeast RNAinteracting with different metal charged IDA Chelating Sepharose matrixin 10 mM HEPES with 250 mM NaCl at pH 7.0. The metal ions charged wereCu (II), Ni (II), Zn (II), and Co (II), to show the different affinitiesof each metal chelate toward bakers yeast RNA;

FIG. 2 graphs equilibrium adsorption isotherms for bakers yeast RNA onChelating Sepharose charged with various metals with 250 mM NaCl in 20mM HEPES at pH 7.0;

FIG. 3 graphs isotherms of 20-mer homopolymer oligonucleotides showingthe different affinities of each base (A, G, C, and T) toward a Ni (II)charged IDA Chelating Sepharose matrix in 10 mM HEPES with 250 mM NaClat pH 7.0;

FIG. 4 graphs equilibrium adsorption isotherms of 20-mer homopolymeroligodeoxynucleotides on Ni(II) charged Chelating Sepharose matrix with250 mM NaCl in 20 mM HEPES at pH 7.0;

FIG. 5 shows repeated Cu IDA stripping of RNA from plasmid. EtBr stained1% agarose gel of Cu (II) charged Chelating Sepharose matrix batchadsorption experiment of alkaline lysed E. coli with plasmidpBGS19luxwt. Lane 1 is the original lysate; Lane 2 is lysate contactedwith non-charged IDA matrix; Lane 3 is the unbound material after asingle batch adsorption; Lane 4 is Lane 3 after exposure to freshmatrix, Lane 5 similarly is Lane 4 after exposure to fresh matrix andLane 6 is Lane 5 after exposure to fresh matrix;

FIG. 6 shows a plasmid separation on Cu (II) charged IMAC. The plasmidpCMV sport .beta. gal was ran over a 20 mL Cu (II) charged IMAC column(1.times.15 cm Amicon FPLC column packed with Chelating Sepharose FastFlow) in a 20 mM HEPES with 250 mM NaCl at pH 7.0 running buffer (nogradient) at a flow rate of 2 mL/minute. The plasmid passed through thecolumn with no hold-up while RNA and other damaged nucleic acids bind tothe IMAC media and were retained on the column longer (isocraticseparation). Nucleic acids were detected by gel electrophoresis and RNAwas not visible on the 0.8 agarose gels;

FIG. 7 shows a FPLC Chelating Sepharose separation of .beta. ribozymeafter compaction precipitation. The FPLC chromatogram traces the bindingof .beta. ribozyme to a 2 mL HyTrap Chelating Sepharose Column (2chelating Sepharose 1 mL columns in series) and the subsequent elution.The ribozyme was loaded in column running buffer and a gradient was runfrom 0 to 1.5 M NH.sub.4Cl;

FIG. 8 shows repeated Cu(II) IDA stripping of RNA from a plasmidDNA-containing alkaline lysate. Ethidium bromide stained 1% agarose gelof Cu(II)-charged Chelating Sepharose batch adsorption of E. colialkaline lysate with plasmid pBGS19luxwt. 1 mL of an IPA-precipitatedalkaline lysate resuspended in 1 mL IMAC running buffer was contactedwith 50 mL of Chelating Sepharose per batch experiment. Lane 1 is theoriginal lysate; Lane 2 is lysate contacted with metal-free IDA matrix;Lane 3 is the unbound material after a single batch adsorption withCu(II)-charged matrix; and, each of Lanes 4-6 is the previous lane afterexposure to fresh matrix;

FIG. 9 shows (Top) FPLC chromatogram of an alkaline lysate containingpCMV sport b gal plasmid DNA run over a 15 mL Cu(II)-charged ChelatingSepharose column. Approximately 30 mL of lysate containing 3.8 mg/mL ofnucleic acid was passed over the column at 1.5 mL/min. (Bottom) 0.8% Egel (Invitrogen) stained with SYBR Gold to visualize each fraction fromthe FPLC run. The fractions on the upper plot correspond to the lanes inthe gel below;

FIG. 10 shows 2% agarose gel stained with SYBR Gold nucleic acid stain(Molecular Probes) of PCR product cleanup. Lane 1 is the 1 kb ladder;Lane 2 is PCR primers (forward and reverse) for the plasmid pCMV sport bgal (Gibco); Lane 3 is an PCR reaction amplifying an .about.800 byfragment of pCMV sport b gal; Lane 4 is the unpurified PCR product ranthrough a Ni (II) charged spin column; Lane 5 is the elution.of the Ni(II) charged spin column from Lane 4 (eluted with 500 mM imidazol incolumn running buffer); Lane 6 is the unpurified PCR product ran througha Cu (II) charged spin column; and Lane 7 is the elution of the Cu (II)charged spin column from Lane 6 (eluted with 500 mM imidazol in columnrunning buffer);

FIG. 11 shows PCR product cleanup by IMAC. Upper) 1.5% agarose gelstained with SYBR Gold nucleic acid stain (Molecular Probes). Lane 1 isan overload (approx. 1300 ng) of Taq PCR reaction amplifying an 800 byfragment of pCMV sport b gal; Lane 2 is a diluted loading (200 ng) ofLane 1; Lane 3 is overload (200 ng) PCR product mixture from Lane 1 runthrough a Cu(II) charged spin column; Lane 4 is a diluted loading (30ng) of Lane 3; Lane 5 is an overload of the elution of the Cu(II)charged spin column from Lane 3-4 with 500 mM imidazole with 250 mM NaClin 20 mM HEPES at pH 7.0; and, Lane 6 is a 2-fold dilution loading ofLane 5. Lower) 1.5% agarose gel stained with SYBR Gold nucleic acidstain (Molecular Probes) the same as the upper panel except Pfupolymerase was used in the PCR reactions;

FIG. 12 shows a HPLC chromatogram of 20-mer oligodeoxynucleotideheteroduplexes bearing the internal mismatches T/C, G/T, A/C, and A/G.The gradient was run over 21 mL (at 1 mL/min) from 0 mM imidazole to 40mM imidazole in 20 mM HEPES with 250 mM NaCl on a 7 mm.times.7 cm TosoHaas Metal Chelating HPLC column charged with Cu(II);

FIGS. 13A&B depict preferred embodiments of an apparatus of thisinvention; and

FIG. 14 depicts another preferred embodiment of an apparatus of thisinvention.

Table A gives preferred ranges for some of the parameters of theinventions

DEFINITIONS

The term “binding” to a IMAC ligand including an immobilized metal atomand/or ion means interacting with the metal atom and/or ion via anychemical and/or physical mechanisms including, without limitation,hydrogen bonding, coordinate bonding, apolar, ionic or covalent bonding,electrostatic interactions, ionic interactions, covalent interactions,mixture or combinations thereof.

The term “non-shielded” means that a purine and/or pyrimidine groups aresufficiently exposed to be able to bind to metal atoms and/or ionsimmobilized in a matrix, i.e., an IMAC matrix. For example, RNA,co-enzyme A, denatured DNA are all examples of molecules that containnon-shielded purine or pyrimidine moieties or groups. On the other hand,duplex DNA or RNA are examples of shielded molecules containing purineor pyrimidine moieties or groups. Thus, the term non-shielded means apurine or pyrimidine moiety or group sufficiently exposed to be able tobind to a metal and/or ion in an IMAC matrix or ligand.

The term “a purine” includes a single purine or a plurality of purines(one or more purines) with the upper limit bound only by the number ofpurines in the molecular components being analyzed, which can be betweenone and millions and millions.

The term “a pyrimidine” includes a single pyrimidine or a plurality ofpyrimidines (one or more pyrimidines) with the upper limit bound only bythe number of purines in the molecular components being analyzed whichcan be between one and millions and millions.

The term “or” includes “and/or” as well as simply or. Thus, purine orpyrimidine means purine and/or pyrimidine.

The term “IMAC matrix” means a molecular component including immobilizedmetal atoms and/or ions capable of binding non-shielded purines orpyrimidines. The matrix generally comprises a polymer having a ligandattached thereto, where the ligand is capable of immobilizing the metalatoms and/or ions.

The term “ligand” generally means a molecule having a site for binding ametal atom or ion. As used herein the ligands are generally chemicallybonded to a substrate so that the binding site can extend into a medium.The ligand, free or bound to a substrate, is then brought in contactwith a desired metal atom or ion or mixture of metal atoms and ions toform an immobilized metal atom or ion bearing (an IMAC) reagent. TheIMAC reagent can then be used to interact with solutions containingmixtures of compounds containing shielded and non-shielded purinesand/or pyrimidines to the purpose of separation, isolation,purification, quantitation, or the like.

The term “IMAC ligand” means a ligand immobilizing a metal atom, ion ormixture thereof, where the metal atom, ion or mixture thereof is capableof binding compounds including a non-shielded purine moiety, anon-shielded pyrimidine moiety or a mixture thereof.

The term “COT” is used to describe the kinetics of hybridization betweentwo nucleic strands in solution and is defined by the product of[nucleic acid].times.(time). Put simply, when the concentration of twocomplementary strands in a solution is high, then it takes a shortertime for hybridization to occur than it does when one or both of thestrands are present at a low concentration.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that a compound containing a non-shieldedpurine or pyrimidine moiety or group such as a single-stranded nucleicacid molecule, e.g., an oligonucleotide or an RNA molecule or a moleculeincluding A, G, C, T or U, have affinity to an IMAC matrix; while acompound that does not contain a non-shielded purine or pyrimidinemoiety or group or easily accessible aromatic nitrogen on a purine orpyrimidine moiety or group, such as double-stranded DNA, RNA, RNA/DNAcomplexes, has little or no affinity to the same IMAC matrix. Thus, theinventors have demonstrated that the affinity of immobilized metalstoward nucleic acid bases allows the use of IMAC in the separation ofdouble strained nucleic acid polymers from single stranded nucleic acidpolymers, the purification of plasmid DNA, RNA, and/or the removal ofnucleotides and primers from PCR reactions.

Although the present invention is primarily directed to nucleic acids(DNA and/or RNA), the present invention enjoys much broader applicationin the fields of biomolecule separation, purification, identification,quantitation, etc., where the target molecule includes a non-shieldedpurine and/or pyrimidine moieties or residue. Such biomolecules include,without limitation, AMP, ATP, GTP, CTP, NAD, Coenzyme A, Hypoxanthine,Xanthine, Orotic acid, Inosine, or any other biomolecule having a purineand/or pyrimidine moiety sufficiently exposed to permit binding to anIMAC reagent of this invention.

The inventors found that purine-containing, single-stranded nucleic acidmolecules, such as oligonucleotides or unstructured RNA molecules, canbe selectively bound by IMAC matrices. The inventors assume that thebinding may be similar to the binding of histidine, which is known tobind to IMAC matrices. The inventors found that soft metals avoidnonspecific interactions with backbone phosphates. (14-16) The inventorshave found that the high, specific affinity of chelated soft metals fornucleic acid bases allows the use of IMAC in the purification of plasmidDNA and RNA, in the removal of contaminants and primers from PCRreaction products, and in the detection of mismatches in DNAheteroduplexes.

The present invention broadly relates to an immobilized metal affinitychromatography (IMAC) column including an IMAC matrix includingimmobilized metal atoms and/or ions capable of binding compoundscontaining non-shielded purine and/or pyrimidine moieties or groups. Thepresent invention also broadly relates to an immobilized metal affinitychromatography (IMAC) instruments including an immobilized metalaffinity chromatography (IMAC) column or absorbent including a matrixhaving immobilized metal atoms or ions capable of binding compoundscontaining a non-shielded purine or pyrimidine moiety or group.

The present invention also broadly relates to a method for separatingdifferent compounds containing a non-shielded purine or pyrimidinemoiety or group by passing a solution containing the different compoundsthrough an immobilized metal affinity chromatography (IMAC) columnincluding a matrix having immobilized metal atoms or ions capable ofbinding compounds containing a non-shielded purine or pyrimidine moietyor group and analyzing the effluent from the column as a function oftime or over a given period of time.

The present invention also broadly relates to a method for purifying asolution containing a major compound containing a non-shielded purine orpyrimidine moiety or group and a minor compound containing anon-shielded purine or pyrimidine moiety or group by passing thesolution through an immobilized metal affinity chromatography (IMAC)column including a matrix having immobilized metal atoms or ions capableof binding compounds containing a non-shielded purine or pyrimidinemoiety or group and analyzing the effluent from the column as a functionof time or over a given period of time. The column can include metalatoms or ions that have a higher or lower binding affinity for the majorverses the minor compound and separation will results, provided that thebinding affinities are indeed different.

The present invention also broadly relates to a method for purifying asolution containing a compound that does not contain a non-shieldedpurine or pyrimidine moiety or group and a compound that does contain anon-shielded purine or pyrimidine moiety or group by passing thesolution through an immobilized metal affinity chromatography (IMAC)column or absorbent including a matrix having immobilized metal atoms orions capable of binding compounds containing a non-shielded purine orpyrimidine moiety or group and analyzing the effluent from the column asa function of time or over a given period of time.

The present invention also provides a method for removing fluorophoretagged nucleotides or nucleosides from a solution involving passing thesolution through an IMAC column capable of either binding to nitrogenatoms of a base residue in the nucleotides or nucleoside, binding toatoms and/or functional groups of the fluorophores or both and removingall non-bound materials from the column. Thus, fluorophore labelednucleotides or oligonucleotides can be removed from the solution.

The present invention also provides a separation technique involving acompound column including an IMAC zone and another separation zone.Because the IMAC technique is relatively insensitive to the ionicstrength of a solution, an IMAC zone can be used to bind compoundsincluding non-shielded purine or pyrimidine moieties or groups such asRNA and other single stranded nucleic acids followed or preceded by azone designed to affect a separation of the other constituents of thesolution based on the other constituents interaction with the stationarymaterial of the other constituents. Thus, an upper disk, region or zoneof a column having a IMAC region will bind the compounds having anon-shielded purine or pyrimidine moiety or group pulling them out ofsolution before a second section, region or zone of column having adifferent stationary phase such as an anion exchange material or HICmaterial. This type of compound column can be made using monolithtechnology and running a small amount of IMAC ligand onto an activatedcolumn and then flooding the rest of the column with one or moreadditional ligands, quenchers or stationary phases.

The present invention also relates to IMAC pre-filters for purifyingsolution containing large quantities of plasmid. Thus, a short columncoated with an IMAC coating to the present invention is used to bind andremove RNA and other contaminants bindable by IMAC (e.g., on a sampleloading loop) before performing more complete purification on largeanion exchange columns, HIC, size exclusion or some other type ofcolumn.

The present invention also relates to a plasmid separation techniqueusing membranes coated with or impregnated with an IMAC matrix whichfilter or prevent free RNA or molecules containing non-shielded purinesor pyrimidines from migrating to the other side of the membranestructure.

The present invention also relates to a magnetic object such as a bead,stirring rod, or the like either coated with an IMAC ligand or where theobject has a porous outer surface to which an IMAC ligand has beenbonded to, deposited thereon or therein. The present invention alsorelates to the use of these magnetic objects to batch-wise purifysamples containing target single stranded nucleic acid sequences such asRNAs, oligonucleotides, or the like where the single stranded nucleicacid sequences bind to the magnetic object, which can then be removedfrom the solution, washed free of contaminates and eluted to recover thesingle stranded nucleic acid sequences. Alternatively, the magnetic IMACobjects can be used to purify double stranded nucleic acid sequences byremoving contaminating single stranded nucleic acid sequence, e.g.,purifying plasmids or the like.

The present invention also relates to metal beads coated with acomposition including an IMAC ligand to batch bind RNA, single strandedDNA, single stranded oligonucleotides or oligonucleosides or the likefrom a solution containing double stranded DNA such as plasmids toclarify the solution, where the bead can be separated from the solutionvia magnets.

The present invention also relates to an assay using IMAC matricesdeposited in wells of microplates or included in a medium comprising thewells of the microplate and then binding a single strandedoligonucleotide having a fluorescent tag to the matrix. The IMAC matrixeither contains a quencher in close proximity to the metal binding sitesor uses the substrate upon which the IMAC is coated as a fluorescentquencher. When a complimentary sequence is added to a well containingthe fluorescent tagged oligonucleotide, the two pair. The pairedsequence is released and the tag fluoresces. This assay represents aninexpensive route to the construction of DNA chip analogs without all ofthe currently needed fancy lasers, scanners, and fluidics systems.

The present invention also relates to an assay using IMAC matricesdeposited in wells of microplates or medium added to the wells of themicroplate or medium added to the wells of the microplate and thenbinding a single-stranded oligonucleotide having a first tag to thematrix. The IMAC matrix either contains a second tag in close proximityto the metal binding sites or associated with the substrate upon whichthe IMAC is coated acts, where the tags interact to produce afluorescent pair. When a complimentary sequence is added to a wellcontaining the fluorescent tagged oligonucleotide, the two pair. Thepaired sequence is released and the fluorescence stops.

When using a matrix, the bound single-stranded fluorophore is physicallyobstructed from the light path. When a complementary sequence is added,the fluorophore goes into solution where it is exposed to light andfluoresces.

The present invention also relates to an assay using substrates coatedwith IMAC so that a separate fluorophore is placed in the wells tocreate a large stoke shift when the fluorescent tagged oligonucleotideis bound and a normal fluorescent stoke shift when the oligonucleotideis not bound to the IMAC ligand.

The present invention also provides a method for separating poly(A)tailed mRNA from eukaryotic cells including the step of passing asolution comprising eukaryotic cell mRNA through an IMAC columnincluding an IMAC matrix or passing the solution over an IMAC matrix orcontacting the solution with magnets coated IMAC particles andseparating poly(A) tailed mRNA from other mRNA, where the poly(A) tailedmRNA elutes from the IMAC matrix last because the poly(A) tail has ahigher affinity for IMAC matrix—the poly(A) tails are usually 50-200bases long.

The present invention also relates to a method for separating denaturednucleic acid sequences having A rich regions from the complementarystrand having T rich regions.

The present invention also relates to a method for separating denaturednucleic acid sequences having C rich regions from the complementarystrand having G rich regions.

The present invention also relates to a method for separating denaturednucleic acid sequences having A-G or A-C rich regions from thecomplementary T-C or T-G rich regions.

The present invention also relates to IMAC ligand or matrices depositedon capillary walls or in solution (e.g., IMAC ligands or matrices bondedto, associated with or deposited on PEG or dendrimer), where the IMACligand or matrix would alter the retention times of compounds havingnon-shielded purine and/or pyrimidine moieties in capillaryelectrophoresis. The present invention also relates to IMAC ligand ormatrices deposited on a surface or in a gel, where the IMAC ligand ormatrix alters retention times of compounds having non-shielded purineand/or pyrimidine moieties in the gel or on the surface, e.g., slab gelelectrophoresis. In each case, unhybridized single strands, or duplexesbearing mismatches, or duplexes bearing overhangs, or partiallysingle-stranded molecules have longer retention times are retained more.This process can also be performed in an on-off fashion (bound, unboundin a single theoretical plate), e.g., in a microtiter well or on IMACbeads.

The present invention also relates to improving mass spectrometryanalysis of complex nucleic acid mixtures by adding a compositionincluding an IMAC compound or matrix capable of binging compounds havingnon-shielded purine or pyrimidine moieties or groups. After binding, theshielded molecules would have their normal mass to charge responses, butthe molecules bindable to the IMAC ligand would have a mass to chargeratio based on the base molecule and number of IMAC ligands associatedwith the molecule. Additionally, if the IMAC MS fragmentation pattern isknown, the bound materials fragmentation pattern can be easilyextracted.

The present invention also relates to afluorophore-bearing-IMAC-functionality molecule used for hybridizationand/or mismatch detection using polarization or quenching/enhancement,also by retention, partitioning, precipitability, non-dialyzability.

Several of these applications will work best with temperature gradientsor at elevated temperatures, or in the presence of denaturants orcompetitive elutants.

The present invention also relates to the use of IMAC ligands ormatrices in the purification of cellular or viral RNA and non-naturalRNA analogs, especially peptide nucleic acids and modified forms used asaptamers. The present inventions also relates of the use of IMAC inconjunction with many of the DNA applications of hydroxyapatite, e.g.,COT curves for complexity of a DNA sample, or genome and for enrichingsequences of interest by subtractive hybridization.

The present invention also relates to cell type recognition,differentiation and identification by separating duplexed nucleic acidsequences from non-duplexed sequences where one set of sequencescomprises the mRNA of one cell type and the other set of sequences arecomplementary mRNA or cDNA derived from the mRNA of a second cell type.Thus, the mRNA form one cell type can be hybridized with thecomplementary mRNA or cDNA of the second cell type. The solution is thenseparated over an IMAC substrate where the IMAC substrate would bindnon-paired mRNAs and/or mismatched paired molecules. The bound materialcan then be extracted from the IMAC substrate and tested to determinespecific differences between the mRNAs of each cell line. This techniquecan be used to determine cite mutations in cancer versus non-cancer celllines or wild-type versus mutant cell lines. Alternatively, thetechnique can be used to look for homologous sequences across specieswhere nucleic acid sequences (mRNA, chopped DNA, or denatured DNA) fromone species are mixed with complementary nucleic acid sequences of thesame type from another species. Separating all hybridized or partiallyhybridized material from all single-stranded material and analyzing thehybridized material. Once separated, the nucleic acid sequence ofinterest can be increased enriched for cloning and/or sequencing,chelated-metal-based nucleic acid stains and dyes, e.g., foridentification of dead (permeable) cells or unhybridized spots in arrays(still single-stranded) chelated-metal-based precipitants.

The present invention also relates to nucleic acid affinityprecipitation using composition having two or more metal chelatingagents binding metals capable of binding compounds having non-shieldedpurine or pyrimidine moieties or groups. Such compositions can be usedto bulk precipitate RNA, single stranded DNA or other molecules havingnon-shielded purine or pyrimidine moieties or groups. The compositionscan include those composition disclosed in U.S. Pat. Nos. 5,310,648 and5,283,339, incorporated herein by reference. Additionally, thecomposition can be any soluble oligomeric or polymeric material thatincludes chelating groups capable of chelating metals with affinitiesfor compounds having purine or pyrimidine moieties or groups.

Additionally, the backbone or spacer groups can be labile (designed tobe chemically and/or physically removable) to such processes as a redoxreaction (redox-cleavable) or photochemically labile to facilitatere-solubilization of the precipitated material. Moreover, functionalizeddendrimers having metal chelating groups are acceptable compositions.

The present invention also related to IMAC chelating agents havingmultiple chelating agents so that multiple metals can be bound to theIMAC matrix. These multimetal IMAC matrices can be used to performseparations or assays based on IMAC affinity for nucleic acids in thepresence of a competitive ligand, such as histidine, etc. to reducenon-specific binding.

The present invention also relates to denaturing IMAC chromatography ofnucleic acid sequences using chemical gradient and/or temperaturegradient and/or other denaturing environments so that the method wouldseparate nucleic acid sequence based on their denaturing characteristicwhich would allow them to bind to the IMAC ligands in the stationaryphase.

The present invention also relates to elution with adenosine, ATP, AMP,GTP or with elution in displacement-chromatography.

The present invention also relates to a method for purifying compositionhaving a non-shielded purine or pyrimidine moiety or group from a crudemixture of materials. This method would be especially useful in thepurification of AIDS drugs such as AZT, DDI or the like. The methodwould also be especially useful in the purification of ribavirin,riboflavin, acyclovir, inosine, s-adenosyl methionine or other naturalproducts or pharmaceuticals that include a non-shielded purine orpyrimidine moiety or group.

The present invention can also be used to strip out nucleotide and/ornucleosides and/or nucleic acid sequences from lysates or othercomposition containing protein and nucleic acid sequences and monomersto derive food stuffs for animal including human consumption using IMACligands or matrices. In addition, the stripping step can be preceded bytreating the lysates or other protein rich compositions with nucleasesor ribonucleases to convert large nucleic acid sequence to smallernucleic acid sequences or monomers. Once the nucleotide or nucleosidesor nucleic acids have been recovered from the IMAC ligands or matrices,the nucleotide or nucleosides or nucleic acids can be separated andpurified to provide a large scale source of nucleotide or nucleosides ornucleic acids.

The present invention also relates to a purification method for nucleicacids involving subjecting a mixture of nucleic acids of a DNase andseparating the resulting fragments.

The present invention also relates to an IMAC column having a ½ Copper(II) ions or a similar ion that binds aromatic nitrogen, imidazol groupsand ½ with an ion like Ti(III) or Cr(II) that prefers ketogroups. Usingsuch mixed metal IMAC columns one can achieve higher affinity forcontaminants (e.g., RNA in plasmid DNA) or improve selectivity.

The present invention relates to substrates having deposited thereon ortherein IMAC ligands or derivatives IMAC ligands, where thederivatization allows the IMAC ligand to bond to reactive groups on thesubstrate surface or within the substrate.

The present invention also relates to methods of separating using anIMAC ligand containing solid material in a fluidized bed configuration,where a solution is force through the material in an upflow conditionresulting in fluidization of the material and the material of interestbinds to the material or the contaminants bind to the material. Thematerial of interest or contaminant can then be released from thematerial using a displacement agent.

The present invention also relates to separating a library ofsingle-stranded nucleic acid sequence having a poly A leader or trailerfrom their complimentary sequences where the sequences where derivedfrom a plasmid which has undergone random mutations in a tissue culture.

An additional preferred embodiment of the IMAC device is the use ofmixtures of metal ions to vary affinity for unshielded purines andpyrimidines. These mixtures could be used to vary affinity for specificsequences since certain metals have high affinity for purines and othershave high affinity for pyrimidines. These mixtures could result in asuperior device for many of the examples stated here-in.

The present invention also relates the separation of PNA (Peptidenucleic acids (PNA) are DNA mimics with a pseudopeptide backbone. PNA isan extremely good structural mimic of DNA (or RNA), and PNA oligomersare able to form very stable duplex structures with Watson-Crickcomplementary DNA, RNA (or PNA) oligomers, and they can also bind totargets in duplex DNA by helix invasion.) via a polypurine (orpolypyrimidine tail) since PNA is not degradable by nucleases, adeoxyribose tail could be used as a tag that would bind an IMAC resinselectively with a high affinity. Then after separation the tail couldbe destroyed by nuclease digestion using DNAse immobilized to a solidsupport or just DNA. It would be fairly simple to separate the PNA fromthe bases left behind.

The present invention also relates to substrates including aconcentration gradient of IMAC ligands bonded thereto, where thegradient extends for a profile of the substrate such as from a frontedge of the substrate to a back edge of the substrate, with slices ofthe substrate at right angle to the gradient has a constant IMAC ligandconcentration. The substrate can be a gel on a gel plate or a columnsubstrate or any other substrate commonly used in gradient gelseparations or other similar analytical separation techniques.

The present invention also relates to the use the gradient IMAC ligandsubstrates to separation and/or focus mixtures of nucleic acidsequences, compounds having a non-shielded purine, a non-shieldedpyrimidine, or mixtures thereof, of the like.

The present invention also relates to imprinting on a surface an IMACligand or a substrate having an IMAC ligand bonded thereto for theconstruction of a patterned surface having IMAC ligand regions and IMACligand free regions. The substrate can be a chip and the IMAC ligandregions can be treated with a probe so that probe association withnucleic acid sequences including a complimentary probe subsequence canbe directly identified by a change in the electrical properties of thecircuit on the chip below an IMAC ligand region from which its probe hasbeen release due to interaction with its complimentary subsequence.

The present invention also relates to the purification ofoligonucleotides prepared using the Merrifield solid phaseoligomerization technique.

The present invention also relates gradient HPLC methods for purifyingcomplex mixtures of solutions containing compounds including anon-shielded purine, a non-shielded pyrimidine, or mixtures thereof andcompounds including a shielded purine, a shielded pyrimidine, ormixtures thereof.

Suitable IMAC ligands for use in this invention include, withoutlimitation, any molecule having a molecular moiety or group capable ofcoordinating or chelating a metal atom or ions. One preferred type ofIMAC ligand comprise molecules having a linear or branched carbohydrylgroup (carbon-containing group) and a head group capable of binding ametal atom or ion having the formula Z—R, where Z is a group capable tobinding a metal atom or ion and R is a linear or branched carbohydrylgroup having between about 1 and about 50 carbon atoms or more orCH(R′)CH.sub.2[OCH(R′)CHJ.sub.n where R′ is H or methyl and n is chosenso that the compound has a molecular weight prior to metallation betweenabout 1500 to about 20,000. Other preferred type of IMAC ligandcomprises molecules including a head group, a linking group and a tailgroup represented by the formula Z—R—Z′, where Z is a head group capableof immobilizing a metal atom or ion, R is as described above, and Z′ isa tail group. The tail or Z′ group can be the same of Z or a groupdesigned to chemically bound to a substrate so that the head group, Z,is tethered from the substrate by the linking group R. Such substratereactive Z′ groups include, without limitation, OH, COOH, COOR″, SH, orother groups capable or reacting with a substrate such as sepharose,agarose, polyacrylamide, or similar substrates, where R″ is the same ordifferent from R, but shares the same definition. Again, R can have from1 to 50 carbon atoms or more. R can be an alkyl group, an alkenyl group,an alkynyl group, an aryl group, an alkaryl group, an aralkyl group, agroup where one or more carbon atoms from been replaced by a hetero atomsuch as N, O, P, S, Si or the like. The head or Z groups can include,without limitation, (a) —N(CH.sub.2COOH).sub.2; (b)—NHCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2NH.sub.2; (c)—NHCH.sub.2CH.sub.2N(CH.sub.2CH.sub.2NH.sub.2).sub.2; (d)—NHCH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2N—H.sub.2; (e)—NHCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2NH.sub.2; (f)NHCH(X)COOH (D- or L-amino acids); (g) —N(CH.sub.2COOH)CH(X)COOH[D- orL-N-(carboxymethyl)amino acids], where X in f and g is H, CH.sub.3,CH(CH.sub.3).sub.2, CH.sub.2CH(CH.sub.3).sub.2,CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2CH.sub.2SCH.sub.3, CH.sub.2OH,CH(OH)CH.sub.3, CH.sub.2SH; CH.sub.2COOH, CH.sub.2CONH.sub.2,CH.sub.2CH.sub.2COOH, CH.sub.2CH.sub.2CONH.sub.2, (CH.sub.2)4NH.sub.2,(CH.sub.2).sub.3NHC(NH.sub.2).sub.2; (h)NHC(O)CH.sub.2N(CH.sub.2COOH)CH.sub.2CH.sub.2N(CH.sub.2COOH).sub.2; (i)—NHC(O)CH.sub.2N(CH.sub.2COOH)CH.sub.2CH.sub.2N(CH.sub.2COOH)CH.sub.2CH.-sub.2N(CH.sub.2COOH).sub.2;(j) —N(CH.sub.2COOH)CH.sub.2CH.sub.2N(CH.sub.2COOH).sub.2. (k)—YNHCH.sub.2CH.sub.2N(CH.sub.2CH.sub.2NH.sub.2).sub.2; (l)—YNHCH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2-NH.sub.2;(m) —YNHCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2NH.sub.2;(n)—YOCH.sub.2CH.sub.2N(CH.sub.2COOH)CH.sub.2CH.sub.2N(CH.sub.2COOH).sub.2; (o) —YOCH[CH.sub.2N(CH.sub.2COOH).sub.2].sub.2 wherein Y in PEG- andPPG-based compounds is NHC(O)CH.sub.2CH.sub.2C(O) and in other compoundsit is C(O) only. The ligands also include those ligands described inU.S. Pat. Nos. 5,310,648 and 5,283,339, incorporated herein byreference, and any soluble oligomeric or polymeric material thatincludes chelating groups capable of chelating metals atoms or theircorresponding ions having an affinity for compounds having anon-shielded purine or pyrimidine moieties or groups, or mixtures orcombination thereof.

Particularly preferred head groups include, without limitations, IDA,iminodiacetic acid, (—N(CH.sub.2COOH).sub.2, NTA, nitrilotriacetic acid,(—CH(COOH)N(CH.sub.2COOH).sub.2), or other amine-carboxylic acidchelating groups.

Suitable IMAC metal atoms or ions for use in this invention include,without limitation, any metal atom or its corresponding ions from thePeriodic Table of Elements capable of binding to a compound containing anon-shielded purine or pyrimidine moiety or group. Preferred metal ionsinclude, without limitation, Cu.sup.2+, Zn.sup.2+, Ni.sup.2+, Fe.sup.3+,Fe.sup.2+, Co.sup.2+, Sc.sup.2+, Ti.sup.2+, V.sup.2+, Cr.sup.2+,Mn.sup.2+, Ca.sup.2+, Cd.sup.2+ or Hg.sup.2+ or mixtures or combinationsthereof. Particularly preferred metal ions include Cu.sup.2+, Zn.sup.2+,Ni.sup.2+, Fe.sup.2+, or Co.sup.2+ or mixtures or combinations thereof.More particularly preferred metal ions include Cu.sup.2+ or Ni.sup.2+ ormixtures or combinations thereof. For metal ions, the preferredcomplexes for attaining the desired ion includes, without limitation,any metal salt that is soluble in the metal charging buffers having acounterion that does not adversely affect the IMAC ligand or thesubstrate to which the ligand is bound. Preferred metal salts includemetal halides such as metal fluorides, metal chlorides, metal bromides,or metal iodides or mixture thereof, metal carboxylates, metalcarbonates or bicarbonates, metal nitrates, metal phosphates, metalsulfates, metal oxychlorides, metal or similar metal complexes.Particularly preferred metal complexes are the metal chlorides.

Suitable substrate upon which the IMAC ligands can be bonded to,attached to or associated with include, without limitation,non-capillary columns, capillary columns, gels, chip surfaces,microplate surfaces, large pore zeolites, mordenites, fugucites or thelike, porous foams, porous resin, polymer beads including macroreticularbeads, surfaces of non-porous monolithic structures such as inorganicmonolithic structures used in catalytic converts or polymeric structuressuch as epoxide resins including CIM monoliths made by BIA Separationsof Ljubljana, Slovenia, or the like.

Suitable polymer substrates for IMAC ligand functionalization, include,without limitation, sepharose, chemically and/or physically modifiedsepharose, agarose, chemically and/or physically modified agarose, otherpolymeric sugars or chemically and/or physically modified versionsthereof, cellulose, chemically and/or physically modified cellulose,polyolefins, chemically and/or physically modified polyolefins,polydienes, chemically and/or physically modified derivative polydienes,polyurethanes, chemically and/or physically modified polyurethanes,polypeptides, chemically and/or physically modified polypeptides,polyamides, chemically and/or physically modified polyimides,polyalkyleneoxides, chemically and/or physically modifiedpolyalkyleneoxides such as polyethyleneglycols, chemically and/orphysically modified polyethyleneglycols, silicones, elastomers,thermoplastics, thermoplastic elastomers, or any other polymericsubstrate.

Suitable membranes include, without limitation, impermeable membranes,permeable membranes or semi-permeable membranes or chemically orphysically modified membranes.

Suitable inorganic supports include, without limitation, silicas,silicates, aluminas, silca-aluminas, zeolites, mordenties, fugasites,aluminates, clays, monoliths, honeycombed monoliths, or any otherinorganic support or chemically or physically modified supports.

Suitable metallic supports include, without limitation, any metal suchas gold, gold alloys, platinum, platinum alloys, silver, silver alloys,iron, iron alloys such any steel, copper, copper alloys such as brass orbronze, tin and tin alloys, aluminum and aluminum alloys, silicon andsilicon alloys, other semiconductors, or the like or chemically orphysically modified metallic supports.

Suitable electronic chip include, without limitation, any electronicdevices having a surface capable of being chemically functionalized.

Suitable chemical and physical modification processes includes, withoutlimitation, chemical functionalization with reactive chemical agents,ion and/or atom bombardment and/or implantation, reactive extrusion,chemical etching, chemical deposition, or any other chemical or physicalmodification that permits IMAC ligands to be bounded to a substrate.

DETAILED DESCRIPTION OF EXPERIMENTS OF THIS INVENTION

Protocols for Nucleic Acid Separation

Spin Column Separation Technique

One preferred method for practicing this invention is the so-called SpinColumn Separation technique. This technique is useful for PCR productseparation using nickel charged columns and possibly for plasmidmini-preps using copper charged columns. The techniques uses thefollowing materials: (o) Chelating Sepharose Fast Flow, AmershamPharmacia Biotech; (p) Snap on spin columns for microcentrifuge tubesfrom Promega; (q) Imidazol; (r) Ammonium Chloride; (s) Sodium Chloride;(t) HEPES; and (u) a metal ion source such as CuCl.sub.2, NiCl.sub.2,and ZnCl.sub.2.

Spin Columns Preparation

This section describes the general procedure for preparing spin columnsfor subsequent use.

Promega mini-prep microfuge snap-on columns with Chelating Sepharosemedia from Pharmacia biotech were used. Load media in 70% EtOH wasdirected onto the mini-columns. Generally, 50 uL of load media was usedper column; however, the amount of load medium can be reduced based onamount of nucleic acid in samples. The columns were then spun for.about.1 minute at maximum speed in a microcentrifuge. Two 150 uLvolumes of water were used to wash the columns. Two 150 uL volumes of asolution of 50 mM X.sup.+2Cl.sub.2.sup.-1 in DI water was used tometallate the columns. The metallated columns were then washed with two150 uL volumes of IMAC running buffer (250 mM NaCl, 20 mM HEPES at pH7.0). At this point, the column are charged and ready to bind nucleicacids. The columns are then switched to clean microcentrifuge tubes forsubsequent use.

Spin Column Binding

This section describes the general procedure for binding materials ofinterest to spin columns prepared as described above.

For lysate samples, the lysate is resuspended in IMAC running buffer andloaded directly onto the IMAC spin column. The column is then spun forabout 1 minute at maximum speed in a microcentrifuge. For samples fromDNA and/or RNA sequencer or PCR reactions or other sequencing protocols,the sample or reaction product is loaded directly onto a metal chargedspin column, preferably a nickel charged spin column and centrifuge. Theprimers and bases bind to the spin column and the purified sequencingproduct passes through the column and can be recovered from thesupernatant in the microfuge tube. For plasmid purification, the sampleis loaded directly on to a metal charged spin column, preferably acopper charged spin column and centrifuge. In the case of plasmid DNA,the RNA and other contaminants bind to the spin column and the clarifiedplasmid can be recovered from the supernatant in the microfuge tube. Thespin columns can then be eluted with EDTA and recharged or reuse.

Spin Column Elution

This section describes the general procedure used to elute a used spincolumn for recharging and reuse.

Generally, elution is accomplished by added 150 .mu.L of an elutionbuffer to the spin column in the microfuge tube and centrifuging thecolumn for .about.1 minute at maximum speed on an Eppendorfmicrocentrifuge. There are several elutants that can be used including,without limitation; imidazole, ammonium chloride, histidine, etc. Thechoice of elutant will generally depend on the type of IMAC separationthat was used. The elutant should be compatible with IMAC separations.The overall preferred elutant is imidazole. The elutant buffer generallyis a 200 mM imidazole IMAC running buffer. The elution results in a pHshift from a pH 7 for the running buffer to a pH 3.5 for the elutionbuffer. An EDTA solution can also be used to strip the metal ions fromthe matrix.

The choice of metal used to charge the spin columns depends on theapplication. Generally, Cu.sup.2+ charged spin column have the highestloading capacity, but is known to strongly bind even one exposedhistidine in proteins. The inventors have confirmed that Cu.sup.2+ alsostrongly binds biomolecules having even a single non-shielded purine orpyrimidine moiety. Thus, Cu.sup.2+ may not be the metal of choice inseparation applications—shows reduced discrimination between differentmolecules, but may be the metal of choice in purification applicationsdesigned to remove either double stranded nucleic acid sequences fromsingle stranded impurities or vis-a-versa. On the other hand, Ni.sup.2+and Zn.sup.2+ show modest binding capacities, but are known to bindproteins with two or more adjacent exposed histidines. Thus, Ni.sup.2+and Zn.sup.2+ may represent the metals of choice in separationapplications—show increased discrimination between different molecules,but may be less desirably in purification applications.

In this invention, spin columns were loaded with Cu.sup.2+, Ni.sup.2+and Zn.sup.2+ capacities of 6, 4, and 1 mg/mL matrix, respectively.There are relationships between selectivity and loading capacity thatappear to affect separation and binding efficiencies as described morefully herein and are apparent from the data and allow for theformulation of separation protocols and methods for different systems.

Batch Binding

Another preferred method for practicing this invention is the so-calledBatch Binding technique described below.

Media Preparation

Chelating Sepharose Fast Flow adsorbent (Amersham Pharmacia Biotech) wascharged before isotherms were run. The Sepharose Matrix was pippetedinto a 2.2 mL microcentrifuge tube and centrifuged at maximum speed inan Eppendorf microcentrifuge. After centrifugation, the supernatant wasremoved by decantation. Next, 0.5 mL of distilled water was added,vortexed, and decanted. This buffer wash was repeated 3 times. Next, 1mL of a 50 mM solution of CuCl.sub.2, CoCl.sub.2, ZnCl.sub.2, orNiCl.sub.2 was added to the tube, vortexed, centrifuged, and decanted.This charging step was repeated 3 times to insure complete loading ofthe matrix. Next, the charged media was washed with IMAC binding buffer(20 mM HEPES containing 250 mM NaCl at pH 7.0), this wash was repeatedtwice. Finally, ½ of the matrix volume of IMAC buffer was added to thetube to make a slurry.

Binding

Binding is performed by adding slurried IMAC matrix, as prepared above,directly to a sample in an appropriate buffer. Although any buffer canbe used, preferred appropriate buffers include any buffer that does notcause precipitates to form or cause lose of metal ions bound to the IMACmatrix. Therefore, the buffer preferably should not contain EDTA or asimilar soluble metal chelating agent. The sample to which the slurriedIMAC matrices can be added include, without limitation, PCR products,Sanger Sequencing reaction products, cell lysates, impure plasmidproducts, impure pharmaceuticals, impure RNA products, impureRNA-enzymes, or any other product including impurities having pairedpurines and/or pyrimidines or impurities having non-shielded purineand/or pyrimidines. The sample to which the slurried IMAC matrix hasbeen added, can then be agitated if necessary and centrifuged after asufficient time for binding.

Elution

Once binding is completed, elution can be performed in a manner similarto the elution step described in the spin column elution section above.The elution buffer can be chosen to fit the needs of the separation.Generally, to elute, the elution buffer is added to the recovered IMACmatrix from the binding section above. The solution can then be agitatedor vortexed and centrifuged and the supernatant removed by decantation.The process can be repeated several time to ensure complete elution.

Plasmid DNA Separations Using IMAC

Another preferred method for practicing this invention is in thepurification of plasmid DNA. This method generally entails the use ofthe following additional materials: (1) an FPLC System or similar fastflow preparative liquid chromatography system and (2) an HPLC column,where the column is either prepacked or packed before use with asubstrate having IMAC ligands attached thereto such as a chelatingSepharose substrate, a chelating agarose substrate, a chelatingpolyacrylamide substrate, or other similar substrates modified with anIMAC ligand.

Plasmid Lysate Clarification:

Plasmid lysates were clarified by column chromatography using an FPLCsystem, such as FPLC systems available from Amersham Pharmacia Biotech.Two, 1 mL HyTrap chelating columns connected in series were used toseparate/purify a sample containing pCMV Sport b gal plasmid DNA (GibcoBRL, 7.9 kb) and cell lysate impurities. The plasmid samples wereprepared by solution-phase compaction precipitation as described inMurphy, et. Al., Nature Biotech., August 1999, incorporated herein byreference.

The HyTrap columns were first equilibrated with 10 column volumes ofdeionized H.sub.2O, followed by a 50 mM M.sup.2+Cl.sub.2 solution, whereM.sup.2+ is a metal ion, preferably, Cu.sup.2+, Zn.sup.2+, Ni.sup.2+,Fe.sup.3+, Fe.sup.2+, Co.sup.2+, Ca.sup.2+, Cd.sup.2+ or Hg.sup.2+ ormixtures or combinations thereof. The inventor found that the preferredmetal ion for plasmid lysate clarification was Cu.sup.2+. The Cu.sup.2+solution was continuously applied to the column until completesaturation was observed by 254 nm ABS measurement. Next, the columnswere washed with 2 volumes of deionized water followed by equilibrationwith 10 column volumes of 10 mM HEPES, 250 mM NaCl, which is thepreferred column running buffer. Plasmid samples were loaded directlyonto the column. The column effluent containing the purified plasmid DNAwas collected in plastic fraction tubes. After separation, columns wereregenerated by first running 10 mM EDTA over the column, then a solutionof 1 N NaOH and then 3 M NaCl.

The plasmid samples used in these examples were prepared using thesolution-phase compaction precipitation method, which produces a lysatehaving >95% plasmid DNA and relatively low endotoxin levels. However,the present method of plasmid DNA purification can be used for anyimpure plasmid sample regardless of its manner of preparation.

RNA Separation Using IMAC

Another preferred method of this invention is theseparation/purification of RNA. This technique also generally entailsthe use of the following additional materials: (1) an FPLC System orsimilar fast flow preparative liquid chromatography system and (2) anHPLC column, where the column is either prepacked or packed before usewith a substrate having IMAC ligands attached thereto such as achelating Sepharose substrate, a chelating agarose substrate, achelating polyacrylamide substrate, or other similar substrates modifiedwith an IMAC ligand. Preferably, HyTrap Chelating Sepharose columns fromPharmacia or Amicon columns packed with Chelating Sepharose Flow havingan aspect ratio of 10 are used for RNA separation/purification.

RNA Separation

RNA was purified by column chromatography using an FPLC system such asthe FPLC systems available from Amersham Pharmacia Biotech. Two, 1 mLHyTrap chelating columns connected in series were used toseparate/purify samples including a b ribozyme, an E. coli expressedribozyme having 83 bases, and cell lysate impurities.

The HyTrap columns were first equilibrated with 10 column volumes ofdeionized H.sub.20, followed by a 50 mM M.sup.2+Cl.sub.2 solution, whereM.sup.2+ is a metal ion, preferably, Cu.sup.2+, Zn.sup.2+, Ni.sup.2+,Fe.sup.3+, Fe.sup.2+, Co.sup.2+, Ca.sup.2+, Cd.sup.2+ or Hg.sup.2+ ormixtures or combinations thereof. As with plasmid samples, the inventorsfound that the preferred metal ion for RNA lysate clarification was alsoCu.sup.2+. The Cu.sup.2+ solution was continuously applied to thecolumns until complete saturation was observed—appearance of metal ionsin the column effluent as measured using ABS measurement as 254 nm. Thecolumns were washed with 2 volumes of deionized water and equilibratedwith 10 column volumes of 10 mM HEPES, 250 mM NaCl, the preferred columnrunning buffer.

Next, an RNA sample was loaded onto the column. Separation/purificationwas achieved by running a buffer gradient, where the gradient was columnrunning buffer with 0 to 2 M NH.sub.4Cl therein. The ribozyme was elutedover this range of the NH.sub.4Cl gradient and collected in plasticfraction tubes. After separation, columns were regenerated by firstrunning 10 mM EDTA over the column, then a solution of 1 N NaOH and then3 M NaCl.

The present invention is also applicable to the separation, purificationand/or isolation of other types of RNA such as tRNA, mRNA, ribosomalRNA, other RNA containing enzymes, co-factors containing purine and/orpyrimidine moieties, co-enzymes containing purine and/or pyrimidinemoieties, any bio or synthetic molecule containing an unshielded U, T,G, C or A, or the like.

Other Applications of IMAC with Nucleic Acids:

The present invention is also well-suited for use in single nucleotidepolymorphism (SNP) detection. A preferred method of this inventioninvolves using an HPLC IMAC column to detected SNPs, where thispreferred methods has the advantage of exposing the SNP containingsample to a sufficiently large, preferably, maximum, number oftheoretical separation plates, yet maintaining an acceptable separationspeed. Other applications for which the present invention is ideallysuited include, without limitation, the purification of RT PCRreactions, purification, separation or identification of DNA sequencingreaction products, separation of labeled oligonucleotides, separation ofmixture of oligonucleotides, or the like.

In addition, the metal ion chelating ligands (IMAC ligands) such as theIDA ligand, the preferred ligand in Chelating Sepharose and most otherIMAC media, has been derivatized and attached to other substratesincluding polyacrylamide, derivatized polyacrylamide, agarose,derivatized agarose or other substrates. These other substrates to whichan IMAC ligand is attached, allow the IMAC method of this invention tobe extended to electrophoretic and membranes separation techniques.These alternate metal immobilized substrates are also useful for nucleicseparation providing interesting analytical possibilities forderivatized polyacrylamide gels (SNPs, etc.) and faster nucleic acidseparations using columns or membranes. Thus, the IMAC analyticaltechniques of this invention are adaptable to running IMAC modified SNPHPLC columns to separate nucleotides and other nucleic acid containingmolecules. The techniques are even applicable to attaining sequenceinformation based on the observed differential IMAC matrix bindingaffinities of the different bases: A>G>C.gtoreq.T.

Materials and Methods

Nucleic Acids Utilized

Plasmids used were pBGS19luxwt (Genbank #, 6 kb) and pCMVsportbgal(Gibco BRL, 7.9 kb). Total bakers yeast RNA (Sigma) was used for RNAbinding studies. 20-mer oligodeoxynucleotides homopolymers were obtainedfrom MWG Scientific. In addition, an E. coli-expressed ribozyme (86bases) (18) was used in RNA binding experiments as a target molecule.

Batch Equilibrium Isotherms 1

Equilibrium adsorption isotherms were measured, in duplicate, in 1.9 mLmicrocentrifuge tubes (Fisher Scientific). Chelating Sepharose Fast Flowadsorbent (Amersham Pharmacia Biotech) was charged before isotherms wererun. Matrix was pippeted into a 2.2 mL microcentrifuge tube, centrifugedat maximum speed in an Eppendorf microcentrifuge, and the supernatantwas then decanted. Next, 0.5 mL of distilled water was added, vortexed,decanted as listed above, and then repeated twice. Then, 1 mL of 50 mMmetal ion solution (either CuCl.sub.2, CoCl.sub.2, ZnCl.sub.2, orNiCl.sub.2) was added to the tube, vortexed, centrifuged, decanted, andthen repeated twice to ensure complete loading of the matrix. Next, thecharged media was washed with IMAC binding buffer (20 mM HEPES with 250mM NaCl at pH 7.0) and repeated twice. The final step was to add a ½volume of IMAC binding buffer to create a slurry.

In tube preparation for isotherm measurement, the following order ofaddition was followed: 20 mM HEPES (Sigma) pH 7.0 with 250 mM NaCl,nucleic acid dissolved in IMAC binding buffer, and 20 ml 50% by volumeChelating Sepharose adsorbent in IMAC binding buffer.

After vortexing, tubes were rotated end-over-end in a Roto-Torque HeavyDuty Rotator (Cole-Palmer Instrument Co.) for 10 minutes. Afterequilibration, the tubes were centrifuged in an Eppendorfmicrocentrifuge for 2 minutes and the supernatant was removed fornucleic acid concentration measurement by absorbance at 260 nm.

Batch Equilibrium Isotherms 2

Equilibrium adsorption isotherms were measured, in duplicate, inmicrocentrifuge tubes (Fisher Scientific). Chelating Sepharose Fast Flowadsorbent (Amersham Pharmacia Biotech) was charged with metal asfollows: Chelating Sepharose was pippeted into a 1.9 mL microcentrifugetube, repeatedly washed by the addition of distilled water, centrifugedand decanted. The matrix was loaded by 3 cycles of addition of 1 mL of50 mM metal chloride solution, then vortexing, centrifugation, anddecantation. Next, the charged adsorbent was washed three times withIMAC binding buffer (20 mM HEPES (Sigma) with 250 mM NaCl at pH 7.0).The final step was to add one volume of IMAC binding buffer to create aslurry.

For isotherm measurement the following order of addition was followed:IMAC binding buffer, nucleic acid dissolved in IMAC binding buffer, and20 ml 50% by volume adsorbent in IMAC binding buffer.

After vortexing, tubes were rotated end-over-end in a Roto-Torque HeavyDuty Rotator (Cole-Palmer) for 10 minutes, a time found sufficient forequilibration in control experiments. After equilibration, the tubeswere centrifuged in an Eppendorf microcentrifuge for 2 minutes and thesupernatant was removed for nucleic acid concentration measurement byabsorbance at 260 nm.

Homopolymer Isotherms 1

The oligonucleotides used were 20-mer homopolymers of A, G, T, and C.Isotherms were performed as detailed above except the total volume was200 mL and the tubes were eluted with 385 mL of 500 mM imidazol in IMACbinding buffer (15 mL of the supernatant was left in solution). Inaddition, 4 mL of Chelating Sepharose Fast Flow was used charged with Ni(II) to perform these isotherms.

Homopolymer Isotherms 2

Homopolymer isotherms were measured as detailed above except in 0.6 mLmicrocentrifuge tubes with a total volume of 200 ml, containing 4 ml ofChelating Sepharose Fast Flow. The tubes were eluted with 385 ml of 500mM imidazole in IMAC binding buffer (15 ml of the original supernatantwas left in solution making the actual volume 400 ml).

Plasmid Lysate Clarification 1

Plasmid lysates were clarified by column chromatography using an FPLCsystem (Amersham Pharmacia Biotech). A 20 mL Amicon FPLC column (1cm.times.15 cm bed height) was used along with pCMVsportbgal plasmid DNAcontaining cell alkaline cell lysates. Columns were first equilibratedwith 10 column volumes of DI H.sub.20. Next, 50 mM metal ion solution(chloride salt) was applied to the columns until complete saturation wasobserved. Then, the column was washed with 2 volumes of water and thenequilibrated in IMAC binding buffer with 10 column volumes. Plasmidsamples were loaded directly onto the column. After separation, columnswere regenerated by first running 10 mM EDTA over the column, then asolution of 1 N NaOH and 3 M NaCl.

Plasmid Lysate Clarification 2

Plasmid lysates were clarified by column chromatography using an FPLCsystem (Amersham Pharmacia Biotech) and a 20 mL Amicon FPLC column (1cm.times.15 cm). Columns were first equilibrated with 10 column volumesof deionized water, and 50 mM metal chloride solution as applied to thecolumn until complete saturation was observed by monitoring absorbanceat 254 nm. The column was then washed with 2 volumes of water andequilibrated in IMAC binding buffer over 10 column volumes. Plasmidsamples were loaded directly onto the column. After separation, columnswere regenerated with 50 mM EDTA at pH 8.0 and then a solution of 1 MNaCl in 1 N NaOH.

PCR Reaction Cleanup 1

PCR reactions were run on a Perkin-Elmer GeneAmp 2400 PCR system using(for a 100 mL reaction) 3 units of Promega Taq polymerase, 10 mL Promega10.times. reaction buffer with MgCl.sub.2, and Promega total dNTPs. Theselected target was a 7 Kb plasmid 0.25 mg/100 mL reaction pCMV sport bgal (Gibco) with the forward and reverse PCR primers (1 mM of eachprimer/100 mL reaction) SEQ. ID NO. 1 5′ TAATTGTTGCCGGGAAGCTAGAG 3′ andSEQ. ID NO. 2 5′ TCGCATTGAATTATGTGCTGTGTAG 3′(MWG Biotech), whichamplified an 800 base region of the plasmid DNA. The PCR was run for 25cycles at the following temperatures: Denaturing at 94.degree. C. (5.5min), primer annealing at 55.degree. C. (0.5 min), and base extension at72.degree. C. (7.5 min).

Promega mini-prep microfuge snap on columns with Chelating Sepharosemedia from Pharmacia biotech were used to make spin columns and on eachsnap on column 100 mL of media (in 70% EtOH) was loaded directly ontothe mini-column Then columns were centrifuged for .about.1 minute at maxspeed in an Eppendorf microcentrifuge. Next, water (150 mL), metal ionsolution (50 mM metal ion X (II) in DI water, 150 mL), and wash withIMAC running buffer (250 mM NaCl, 20 mM HEPES at pH 7.0, 150 mL) was runover the column twice. At this point, the column was charged and readyto bind nucleic acids. The columns were then switched to cleanmicrocentrifuge tubes and the PCR reaction was loaded directly onto thecolumn.

PCR Reaction Cleanup 1

PCR reactions (100 mL) were run on a Perkin-Elmer GeneAmp 2400 PCRsystem using 2 units of Taq polymerase, 10 mL of 10.times. reactionbuffer with MgCl2, and 800 mM dNTPs (all from Promega). The target was40 pg/reaction of pCMV sport b gal (Gibco) with the forward and reversePCR primers (0.1 nmol each) SEQ. ID NO. 3 5′ TAA TTG TTG CCG GGA AGC TAGAG 3′ and SEQ. ID NO. 4 5′ TCG CAT TGA ATT ATG TGC TGT GTA G 3′ (MWGBiotech), which amplified an 800 base region of the plasmid DNA encodingbeta-lactamase. The PCR was run for 25 cycles of denaturation at 94 ffC(45 seconds), annealing at 55 ffC (30 seconds), and extension at 72 ffC(3 minutes).

Promega mini-prep microfuge snap-on columns filled with 100 mL ChelatingSepharose were used as spin columns. The columns were washed with water,charged with metal chloride solution (50 mM metal chloride in DI water),and finally washed with IMAC running buffer. The columns were thenswitched to clean microcentrifuge tubes and the PCR reactions wereapplied directly onto the spin columns.

Mismatch Detection

Duplexes of oligodeoxynucleotides with internal, single-base mismatcheswere all based on the same 50% G/C 20-mer oligonucleotide SEQ. ID NO. 5(5′ CAG ACG ATA GTC CTA GTT GC 3′) and its complement. Mismatchdetection used a 7 mm.times.7 cm Toso Haas HPLC chelating column run at1 mL/min on a Waters 600E HPLC system.

Oligonucleotides were resuspended in IMAC running buffer, allowed tohybridize at 42 ffC for 10 minutes, and loaded via a 400 mL loop.Elution was by four column volumes of IMAC running buffer and a gradientfrom 0 to 14 mM imidazole in IMAC running buffer. The column wasregenerated by 10 mM EDTA followed by a solution of 3 M NaCl in 0.1 NNaOH.

EXAMPLES

Equilibrium Adsorption Isotherms 1

To test binding capacities and trends, equilibrium adsorption isothermswere run to find affinities of RNA and DNA to IMAC matrices. The firstexperiment ran was to distinguish the different binding affinities ofRNA and double-stranded DNA. In the example, equilibrium isotherms wereperformed using Zn (II) charged IDA IMAC resin using total bakers yeastRNA and pBGS19Luxwt plasmid DNA. The resulting isotherms showed noapparent binding of the plasmid DNA, but a defined binding isotherm forbakers yeast RNA. This experiment represented was the first evidence ofdifferential binding affinity to an IMAC reagent for single-stranded RNAand double-stranded DNA.

Next, binding isotherms were run using different divalent metal ions(Cu.sup.2+, Ni.sup.2+, Zn.sup.2+ and Co.sup.2+) and total bakers yeastRNA to determine the relative binding efficiencies. FIG. 1 shows theisotherms for each divalent metal ion. From the isotherms is it apparentthat each metal has a unique isotherm plateau, i.e., the plateau foreach metal ion occurs at a distinct value of bound material. Thisplateau value corresponds to the loading capacity of the matrix. The RNAbinding efficiencies for each metal For copper, nickel, zinc, and cobaltcharged IMAC matrix are 5, 3.5, 1 and 0 mgs, respectively. RNA capacityof Cu (II) charged IDA-Sepharose was confirmed by breakthrough curveanalysis (not shown). The trends in these isotherms do not follow themetal ion's positions on the periodic chart, but do follow the knowntrends seen for protein binding behavior to IMAC matrices. Cu (II) isknown to bind a single exposed histidine residue; Zn (II) and Ni (II)bind at a minimum of 2 adjacent exposed histidine residues; and, Co (II)is even more selective for a 6 his tag.

The different metals have different affinities for nucleic acids. Eachmetal has a different trade-off between loading capacity andselectivity. Cu (II) has a higher affinity for nucleic acids, while Ni(II) has a higher selectivity for nucleic acid components. Forapplications where a very high binding affinity is desired (plasmidseparation, etc.), Cu (II) is the metal of choice. However, if gradientsare run to get a specific product from a mixture (RNA separations, PCRproduct cleanup), selectivity is more important and Ni (II) and/or Zn(II) should be considered.

Equilibrium Adsorption Isotherms 2

Preliminary experiments using Zn(II)-charged IDA-Sepharose with totalbakers yeast RNA and pBGS19luxwt plasmid DNA showed considerable bindingof RNA, but no apparent binding of the plasmid DNA.

The adsorption of RNA on IDA-Sepharose loaded with various metal ions isillustrated in FIG. 2. The amount of RNA bound per mL of IMAC matrix forcopper, nickel, zinc, and cobalt are 5, 3.5, 1 and 0 mg, respectively.The dynamic RNA capacity of Cu(II)-charged IDA-Sepharose was found bybreakthrough curve analysis (at .about.100 cm/hr flow rate) to be 5-7mg/mL (not shown). The metals' RNA affinities follow the establishedtrend of IMAC protein binding behavior.

Binding of 20-Mer DNA Homopolymers 1

To further explore the mechanism by which IMAC matrices bind nucleicacids, oligonucleotides were obtained, which were 20-mer homopolymers ofA, G, T, and C on a deoxyribonucleic acid backbone. Isotherms wereperformed using Ni (II) charged IMAC resin and the resulting curves areshown in FIG. 3. Poly (A) bound with the highest affinity, while poly(G) had an approximately 10 times binding efficiency or selectivity.Poly (C) had a slightly higher affinity for IMAC matrix than that ofpoly (T). In addition, duplexes of the A and T homopolymers and duplexesof th G and C homopolymers had no binding affinity to the IMAC matrixcharged with Ni (II).

Langmuir fits were performed on these homopolymer isotherms andq.sub.max/K.sub.d was plotted. The q.sub.max/K.sub.d of poly (A) isapproximately 10 times higher than that of poly (G) and theq.sub.max/K.sub.d of poly (C) is roughly 5 times higher than that ofpoly (T). Generally, these trends are thought to result form thestructure of the nucleic acid bases. Adenine lacks a hydrogen atom atthe nitrogen atom at position 1 and has a ½ character aromaticitythrough resonance. Guanine has an amine at position 2 compared to ahydrogen atom in the case of adenine, potentially adding an additionalsteric hindrance to the binding of metal ions to the position 2nitrogen. A similar structural difference is seen in the purines wherethymine has a hydrogen atom at position 3 that could lower the affinityof thymine compared to cytosine.

These isotherms lead to some guideline when using IMAC matrices to bindnucleic acids. First, the IMAC charged matrix does not appear to bindthe phosphate backbone or the sugar moieties of the nucleic acidoligomers or polymers. The low affinity of Ni (II) charged IMAC resintoward poly (T) homo-polymer is clear evidence that the binding is notpurely a backbone effect, since poly (A) and poly (G) showed relativelylarge binding affinities for the IMAC matrix. Moreover, because duplexednucleic acid oligomers or polymers did not bind to the IMAC matrix, thebinding does not occur at either the phosphate backbone structure or thesugar moieties. Furthermore, because single-stranded oligonucleotidesbind to the IMAC matrix, but the duplexes of the A/T and G/Chomopolymers do not, the interaction appears to be based on affinity ofthe IMAC matrix toward the actual exposed bases on the single-strandednucleic acids (or single stranded regions of larger nucleic acidmolecules).

Second, the separation appears to be dependent on the number of singlestranded purines available for interaction with the IMAC matrix. Withthe purines having such a large binding capacity, the method can be usedto differentiate nucleic acid sequence with different purine topyrimidine contents, especially at the extremes of purine and pyrimidinecontent. Thus, the higher the single-stranded purine content, the higherthe affinity for the IMAC matrix.

Binding of 20-Mer DNA Homopolymers 2

The relative IMAC affinities of the nucleic acid bases inpolynucleotides were examined using 20-mer oligodeoxynucleotidehomopolymer isotherms on Ni(II)-charged IDA-Sepharose (FIG. 4). A20bound with the highest affinity while G20 had an affinity approximately10 times lower (based on a Langmuir fit). The affinities of thepyrimidines were much lower than those of the purines (approximately60-and 300-fold lower affinity than A20 for C20 and T20 respectively).In addition, the A20/T20 heteroduplex had no detectable bindingaffinity, eliminating the phosphate backbone as a major source ofadsorption affinity. The relative affinities of the homopolymersconfirmed the nucleotide monomer results of Fanou-Ayi and Vijayalakshmi(11) and Hubert and Porath (12).

Plasmid Purification

Purification of plasmid DNA is an added advantage of IMAC. Previous workon affinity precipitation of DNA by compaction agents (19) allows forthe creation of high purity plasmid preparation without the use ofcolumn chromatography. The major contaminant left in the plasmid DNApurified by compaction precipitation is contaminating RNA and linear DNA(1-5%). The IMAC separation technique of this invention is well-suitedto bind the remaining RNA (the minor component) and DNA fragments tofurther purify large quantities of plasmid DNA on relatively small IMACcolumns

FIG. 5 shows repeated Cu IDA stripping of RNA from plasmid. EtBr stained1% agarose gel of Cu (II) charged Chelating Sepharose matrix batchadsorption experiment of alkaline lysed E. coli with plasmidpBGS19luxwt. Lane 1 is the original lysate; Lane 2 is lysate contactedwith non-charged IDA matrix; Lane 3 is the unbound material after asingle batch adsorption; Lane 4 is Lane 3 after exposure to freshmatrix, Lane 5 similarly is Lane 4 after exposure to fresh matrix andLane 6 is Lane 5 after exposure to fresh matrix.

The next step was to run a lysate over a column. Using compactionprecipitation purified plasmid, column tests were done using a FPLCsystem. FIG. 6 shows the chromatogram of plasmid lysate purificationusing a 20 mL IMAC column charged with Cu (II). The plasmid initiallypasses through the column with no contaminating RNA as determined by gelelectrophoresis and 260/280 ratios. In the end, IMAC worked well as afast and efficient means of stripping RNA from a plasmid containinglysates.

RNA Separation

RNA, purified by compaction precipitation, was separated via IMACcolumns (1 mL Hytrap chelating column). FIG. 7 shows the FPLC trace ofthe separation of the b ribozyme by Cu (II) charged IMAC. The bound RNAwas eluted in the figure by ammonium chloride, but elution can also beaccomplished by using EDTA, pH, imidazole, or histidine. The ability tobind RNA and elute by direct base interaction to the IMAC media leads toa separation that operates on a different principle than anion exchange,hydrophobic interaction, and sizing chromatography.

Plasmid and RNA Separation

The nucleic acid discrimination achieved with IMAC suggests applicationof the method to the purification of plasmid DNA from RNA-rich bacteriallysates FIG. 8 shows repeated Cu(II) IDA stripping of RNA from a plasmidDNA-containing alkaline lysate. Ethidium bromide stained 1% agarose gelof Cu(II)-charged Chelating Sepharose batch adsorption of E. colialkaline lysate with plasmid pBGS19luxwt. 1 mL of an IPA-precipitatedalkaline lysate resuspended in 1 mL IMAC running buffer was contactedwith 50 uL of Chelating Sepharose per batch experiment. Lane 1 is theoriginal lysate; Lane 2 is lysate contacted with metal-free IDA matrix;Lane 3 is the unbound material after a single batch adsorption withCu(II)-charged matrix; and, each of Lanes 4-6 is the previous lane afterexposure to fresh matrix.

IMAC column chromatography was used to strip RNA from an E. colialkaline lysate containing the plasmid pCMV sport b gal. FIG. 9 shows anE. coli alkaline lysate purified using a 20 mL Cu(II)-charged IDASepharose column. The plasmid initially passed through the column withan undetectable amount of contaminating RNA as determined by gelelectrophoresis and 260/280 ratio. Initial RNA breakthrough was observedwhen 1 mg/mL of RNA was bound. Approximately 80% of the contaminatingRNA was still bound at a loading capacity of 5 mg/mL.

Previous work on affinity precipitation of DNA from RNA by compactionagents (19) allows for the preparation of a high purity plasmid DNAwithout the use of column chromatography. The major contaminant in theplasmid DNA purified by compaction precipitation is RNA. IMAC adsorptionhas a high capacity for polishing of compaction precipitated DNA byselective adsorption of the minor RNA component.

RNA (b ribozyme), purified by compaction precipitation (20), wasseparated on a 1 mL Cu(II)-charged Hytrap chelating column (not shown)using an 0 to 2 M ammonium chloride gradient. The ability to bind RNAleads to an IMAC separation that operates on a different principle thananion-exchange, hydrophobic-interaction, boronate, and size-exclusionchromatography, allowing orthogonal separations.

PCR Product Cleanup

This example illustrates the utility of this invention in a model PCRreaction system. PCR product cleanup was performed using IMAC spincolumns directly with PCR products. The PCR reactions were loadeddirectly onto the spin columns allowing for the rapid removal of PCRprimers and stunted fragments. FIG. 10 shows a 2% agarose gel of thecleanup of a PCR product using a Ni (II) charged IMAC spin column. Thespin column mainly left behind the double-stranded PCR product and theplasmid DNA template as seen in lane 4 of FIG. 10. In addition, othermetal ions were evaluated for this application. Cu (II) binds, not onlythe primers and fragments, but also the PCR product itself. Because ofits high affinity for aromatic nitrogens, copper is thought to bind tothe ragged ends commonly left by Taq polymerase. Thus, the presentinvention is also related to the direct binding and elution of the PCRfragments.

PCR Product Purification

PCR reaction mixtures were loaded directly onto Cu(II)-charged, 100 mLspin columns for rapid removal of primers and stunted ormismatch-containing products. The IMAC column captured the primers andthe defective products leaving primarily the double-stranded PCR productand the plasmid DNA template (FIG. 11 top and bottom, Lane 3). Partialelution (FIG. 11 top, Lane 5) shows that the column binds not only theprimers and fragments, but also the Taq PCR product itself, presumablythrough mismatched bulges or the 3′A overhang commonly left by Taqpolymerase. However, processing of a Pfu (21) polymerase PCR reactionproduct mixture amplifying the same sequence gave similar results (FIG.11 bottom, Lane 5), but products of this proofreading polymerase werenot as readily retained on the IMAC column. When the PCR product band inthe elutant lanes of the gels were integrated, 1.6 times as much PCRproduct was retained on the IMAC column when Taq polymerase PCRreactions were purified vs. Pfu PCR reactions. In addition, when theIMAC purified Taq PCR product was sequenced improved fidelity was seenover a non-IMAC purified control sample (results not shown).

Mismatch Detection Using IMAQ

Higher resolution IMAC HPLC can separate mismatch-bearingoligonucleotide heteroduplexes, presumably through interactions withbases in the disordered region. As shown in FIG. 12, retentioncorrelates well with the binding affinities of the homopolymers (FIG.4). Especially with the enhanced resolution of metal affinity capillaryelectrophoresis (22), this mismatch separation could serve as the basisof PCR product cleanup, SNP scoring, or hybridization assays.Fragmentation of a potentially mutated gene followed by heatdenaturation and reannealing in the presence of the corresponding wildtype DNA, could allow efficient DNA sequence confirmation by IMAC in ananalysis similar to “tryptic mapping” of proteins.

CONCLUSIONS

IMAC, a robust and widely used chromatographic process, and nowrepresents an effective nucleic acid separation technique with manyapplications. Metal charged IMAC ligands have affinity forsingle-stranded nucleic acids, in particular exposed nucleic acid basesor other molecules including at least one non-shielded purine orpyrimidine moiety. Through isotherm measurement, the IMAC matrix hasbeen proven to bind to the nucleic acid bases and not the phosphateanion backbone or the ribose in the backbone. The aromatic nitrogens inthe nucleic acid bases are the targets of the metal ion interactionmeaning that purines have a higher affinity than pyrimidines for thecharged IMAC matrix. The determined order of affinities areA>G>>C.gtoreq.T.

The nitrogen containing aromatic bases can be effectively stripped froma bacterial lysate or free solution. One preferred application of IMACdescribed herein is the purification of plasmid DNA by retaining RNAcontaminants. In addition, separation of RNA (b ribozyme),single-stranded nucleic acids, and primers, truncated fragments andbases can be easily be removed from sequencing/PCR reactions.

Chelated soft metal ions interact with exposed bases of nucleic acids,and this interaction can serve as the basis of a variety of preparativeand analytical methods useful in genetic technology. In addition, tothose demonstrated here, further applications may include eukaryotic(poly(A) tailed) mRNA isolation, improvement of the quality andclonability of PCR products, and economical SNP scoring and sequenceconfirmation.

Detailed Description of Apparatuses of this Invention

Referring now to FIGS. 13A&B, two preferred embodiments of separationapparatus of this invention, generally 100, are shown to include acolumn 102 having an inlet 104 adapted to receive a sample (not shown),an outlet 106 and a single zone 108, while the column 102 of FIG. 13Bincludes a first zone 108 and a second zone 110. The single or firstzone 108 includes an IMAC matrix with one or more immobilized metalatoms and/or ions bound thereto, while the second zone includes anothermatrix capable of separating sample components based on a differentchemical and/or physical property such as size, charge, hydrophobicity,hydrophilicity, or any other property. Of course, one of ordinary skillin the art should recognize that the two zones in FIG. 13B can be twodifferent columns connected together in series.

The apparatus 100 is utilized by supplying a sample or sample flow 112to the inlet 104 and analyzing an apparatus effluent 114 leaving theoutlet 106. The sample or sample flow 112 can be a single sample or theoutput of an upstream separation apparatus. Moreover, the apparatus 100can be used in a discrete or continuous mode of operation depending onthe nature of the analysis intended.

Referring now to FIG. 14, a preferred embodiment of an analyticalinstrument apparatus of this invention, generally 200, is shown toinclude a sample input unit 202 in fluid communication via a first fluidconduit 204 with a separation unit 206. The separation unit 204 includesat least one separation apparatus of FIGS. 13A&B. The separation unit204 is in fluid communication via a second fluid conduit 208 with adetector unit 210. The detector unit 210 is adapted to convert one ormore properties of an effluent of the separation unit 206 to a signal.The signal is forwarded to an analyzer unit 212 via an electric conduit214, which places the detector unit 210 in electrical communication withthe analyzer unit 212. The analyzer unit 212 converts the signalproduced by the detector unit 210 into a measurement of the detectedproperty. Of course, the detector unit 210 and the analyzer unit 212 canbe a single unit. Generally, the analyzer unit 212 is a digitalprocessing unit including a memory, a digital processor, output devicessuch as a printer, a CRT, or the like and associated software andhardware for communication, storage, retrieval and human interaction.The sample input unit 202 can be an injector unit for injecting orintroducing a single sample or plug of sample into the separation unit206

The term fluid communication means that fluid is able to flow from oneunit to the other unit in the indicated direction. The term electricalcommunication means that an electric signal travels from one unit toanother unit in the indicated direction.

Modifications

While not to be taken as limiting, the following embodiments are also tobe included within the inventions:

A composition comprising a first compound including immobilized metalatoms and/or ions capable of binding compounds containing a non-shieldedpurine or pyrimidine group and a second compound containing anon-shielded purine or pyrimidine group bound to a portion of the metalatoms and/or ions; wherein the second compound is selected from thegroup of RNA, single stranded DNA, and other molecules having anon-shielded purine and/or pyrimidine moiety or group, or wherein thefirst compound comprises a polymeric material including a plurality ofligands bonded thereto, each ligand immobilizing a metal ion selectedfrom the group consisting of Cu.sup.2+, Zn.sup.2+, Ni.sup.2+, Fe.sup.3+,Fe.sup.2+, Co.sup.2+, Sc.sup.2+, Ti.sup.2+, V.sup.2+, Cr.sup.2+,Mn.sup.2+, Ca.sup.2+, Cd.sup.2+ or Hg.sup.2+ or mixtures or combinationsthereof, or wherein the metal ion selected from the group consisting ofCu.sup.2+, Zn.sup.2+, Ni.sup.2+, Fe.sup.2+, or Co.sup.2+ or mixtures orcombinations thereof.

An immobilized metal affinity chromatography (IMAC) column comprising apacking including immobilized metal atoms and/or ions capable of bindingcompounds containing a non-shielded purine or pyrimidine moiety or groupand a compound containing a non-shielded purine or pyrimidine moiety orgroup bound to a portion of the metal atoms and/or ions wherein thesecond compound is selected from the group of RNA, single stranded DNA,and other molecules having a non-shielded purine and/or pyrimidinemoiety or group; or wherein the first compound comprises a polymericmaterial including a plurality of ligands bonded thereto, each ligandimmobilizing a metal ion selected from the group consisting ofCu.sup.2+, Zn.sup.2+, Ni.sup.2+, Fe.sup.2+, Fe.sup.2+, Co.sup.2+,Sc.sup.2+, Ti.sup.2+, V.sup.2+ Cr.sup.2+, M.sup.2+, Ca.sup.2+, Cd.sup.2+or Hg.sup.2+ or mixtures or combinations thereof; or wherein the metalion selected from the group consisting of Cu.sup.2+, Zn.sup.2+,Ni.sup.2+, Fe.sup.2+, or Co.sup.2+ or mixtures or combinations thereof.

A substrate comprising a plurality of ligands bonded thereto, eachligand immobilizing a metal atom and/or ion capable of binding compoundscontaining a non-shielded purine or pyrimidine moiety or group, and acompound containing a non-shielded purine or pyrimidine moiety or groupbound to a portion of the metal atoms and/or ions wherein the secondcompound is selected from the group of RNA, single stranded DNA, andother molecules having a non-shielded purine and/or pyrimidine moiety orgroup; or wherein the first compound comprises a polymeric materialincluding a plurality of ligands bonded thereto, each ligandimmobilizing a metal ion selected from the group consisting ofCu.sup.2+, Zn.sup.2+, Ni.sup.2+, Fe.sup.3+, Fe.sup.2+, Co.sup.2+,Sc.sup.2+, Ti.sup.2+, V.sup.2+, Cr.sup.2+, Mn.sup.2+, Ca.sup.2+,Cd.sup.2+ or Hg.sup.2+ or mixtures or combinations thereof; or whereinthe metal ion selected from the group consisting of Cu.sup.2+,Zn.sup.2+, Ni.sup.2+, Fe.sup.2+, or Co.sup.2+ or mixtures orcombinations thereof; or wherein the substrate is selected from thegroup consisting of a polymer, a column inner wall, a membrane, aninorganic support, a metallic support, and a surface of an electronicchip.

An apparatus comprising a sample input unit, a separation unit, adetector unit and an analyzer unit wherein the separation unit is a zonecomprising an IMAC matrix including metal atoms, metal ions or mixturesthereof capable of binding compound having a non-shielded purine moiety,pyrimidine moiety or mixture thereof; or wherein the zone is a column;or wherein the separation unit comprises a first zone comprising a IMACmatrix including metal atoms, metal ions or mixtures thereof capable ofbinding compound having a non-shielded purine moiety, pyrimidine moietyor mixture thereof and a second zone comprising a second matrix capableof separating compounds based on at least one chemical or physicalproperty; or wherein the second matrix comprises an anion exchangematerial or an HIC material; wherein the first and second zones are acolumns connected in series; or wherein the first zone comprises a firstportion of a column and the second zone comprises a second portion ofthe column.

An apparatus comprising a substrate having an IMAC ligand coatedthereon, bonded thereto, deposited thereon or deposited therein, wherethe substrate is adapted to remove contaminating compounds including anon-shielded purine moiety, pyrimidine moiety, or mixture thereof fromtarget compounds including a shielded purine moiety, pyrimidine moiety,or mixture thereof wherein the substrate is selected from the groupconsisting of a porous stirrer, a filter, a membrane, an interior wallof a vessel, or mixtures thereof; or wherein the contaminating compoundsare RNA and target compounds are plasmids.

A method for separating compounds comprising the step of

contacting a solution comprising compounds including a non-shieldedpurine or pyrimidine moiety and compounds including a shielded purine orpyrimidine moiety with a solid composition including immobilized metalatoms and/or ions capable of binding compounds containing a non-shieldedpurine or pyrimidine moiety to form a supernatant liquid having areduced amount of compounds including a non-shielded purine orpyrimidine moiety; further comprising the step of:

separating the supernatant liquid from the solid composition; or furthercomprising the steps of:

separating the supernatant liquid from the solid composition and

eluting the compounds including a non-shielded purine or pyrimidinemoiety from the solid composition; or wherein the compounds including anon-shielded purine or pyrimidine moiety comprise a nucleoside, anucleotide, a single stranded nucleic acid oligomer, or a singlestranded nucleic aid polymer and the compounds including a shieldedpurine or pyrimidine moiety comprise double stranded nucleic acidoligomers or double stranded nucleic acid polymers; or wherein thesupernatant liquid comprises compounds including a shielded purine orpyrimidine moiety having less than or equal to 5% by weight compoundsincluding a non-shielded purine or pyrimidine moiety; or wherein thesupernatant liquid comprises compounds including a shielded purine orpyrimidine moiety having less than or equal to 1% by weight compoundsincluding a non-shielded purine or pyrimidine moiety; or wherein thesupernatant liquid comprises compounds including a shielded purine orpyrimidine moiety having less than or equal to 0.01% by weight compoundsincluding a non-shielded purine or pyrimidine moiety.

A method for making multisubstrate columns comprising the step ofrunning a small amount of IMAC ligand onto an activated column and thenflooding the rest of the column with at least one additional ligand orstationary phase.

A method for separating compounds comprising the steps of:

passing a solution comprising a mixture of compounds including anon-shielded purine moiety, a non-shielded pyrimidine moiety or mixturethereof through a column including an IMAC ligand, where the ligand iscapable of differentially binding the compounds; and

collecting purified samples of each compound; or further comprising thestep of:

detecting each compound in an effluent from the column as a function oftime from at least one detectable property associated with eachcompound; and

determining the identity of each compound from the detected properties;or wherein the mixture of compounds comprises poly(A) tailed mRNAsequences and other mRNA sequences from eukaryotic cells, where thepoly(a) mRNA sequences elute after the other mRNA sequences; or whereinthe mixture for compounds comprises denatured nucleic acid sequences,where sequences having A rich regions elute after sequences having Trich regions so that complementary strands can be resolved; or whereinthe mixture for compounds comprises denatured nucleic acid sequences,where sequences having C rich regions elute after sequences having Grich regions so that complementary strands can be resolved; or whereinthe mixture of compounds comprises denatured nucleic acid sequenceshaving A-C, A-G, A-C-G, T-G, T-C and or T-G-C rich regions so that thesequences having thee A-C, A-G, and/or A-C-G rich regions elute aftertheir complementary sequences having T-G, T-C and or T-G-C rich regionsresulting in a resolution of complementary sequences.

A method for purifying food stuffs containing purine and/or pyrimidinemoieties comprising the steps of:

forming a crude food stuff comprising cellular constituents includingdigestable proteins and nucleic acid contaminants including anon-shielded purine moiety, a non-shielded pyrimidine moiety or mixturethereof;

contacting the food stuff with substrate comprising an IMAC ligand,where the substrate binds the nucleic acid contaminants; and

removing the substrate comprising the IMAC ligand having bound theretothe nucleic acid contaminants to form a purified food stuff; furthercomprising the step of

treating the crude food stuff with a DNAse, endo or exo nuclease orother nucleic acid digestion enzyme or agent prior to the contactingstep.

A method for purifying a crude compound containing a non-shielded purineand/or pyrimidine moiety comprising the steps of:

forming a crude mixture comprising a target compound and contaminants;

contacting the crude mixture with an agent including an IMAC ligandcapable of binding to the target compound to form an IMAC ligandcomplex;

separating the complex from the contaminants; and

recovering the compound from the complex; wherein the compound is anAIDs drugs selected from the group consisting of AZT or DDI, co-enzymeA, or mixtures thereof.

An assay comprising the steps of:

contacting a microplate substrate comprising wells coated with acomposition comprising a IMAC-oligonucleotide complex including an IMACligand and a single stranded oligonucleotide having a first molecularand/or atomic tag bound to the IMAC ligand; and

contacting a nucleic acid sequence including a second molecular and/oratomic tag with the IMAC-oligonucleotide complex; and

measuring a change in fluorescence when the nucleic acid sequenceincludes a complimentary subsequence to oligonucleotide due to aninteraction between the first and second molecular and/or atomic tags;wherein the first tag is a fluorophore and the second tag is a quencherfor the fluorophore; or wherein the second tag is a fluorophore and thefirst tag is a quencher for the fluorophore; or 2, wherein the tags fora fluorescent donor-acceptor pair.

An assay comprising the steps of contacting a substrate comprising asurface coated with a composition comprising an IMAC ligand and a firstfluorophore with an oligonucleotide including a second fluorophore andmeasuring an effective Stoke shift such that a large effective Stokeshift signifies oligonucleotide binding to the coated substrate and anormal effective Stoke shift signifies no oligonucleotide binding to thecoated substrate.

REFERENCES

1. Porath, J. (1992) Protein Expr. Purif 3, 263-281. 2. Porath, J.,Carlsson, J., Olsson, I. & Belfrage, G. (1975) Nature 258, 598-599. 3.Alexandratos, S. D., Beauvais, R., Duke, J. R. & Jorgensen, B. S. (1998)J. Appl. Polym. Sci. 68, 1911-1916. 4. Yang, L., Jia, L., Zou, H. &Zhang, Y. (1999) Biomed. Chromatogr. 13, 229-234. 5. Nieba, L.,Nieba-Axmann, S. E., Persson, A., Hamalainen, M., Edebratt, F., Hansson,A., Lidhoim, J., Magnusson, K., Karlsson, A. F. & Pluckthun, A. (1997)Anal. Biochem. 252, 217-228. 6. Holmes, L. D., Serag, A. A., Plunkett,S. D., Todd, R. J. & Arnold, F. H. (1992) Methods: A Companion toMethods in Enzymology 4, 103-108. 7. Van Dam, M. E., Wuenschell, G. E. &Arnold, F. H. (1989) Biotechnol. Appl. Biochem. 11, 492-502. 8. Spiro,T, G. (1980) Nucleic Acid—Metal Ion Interactions (John Wiley, NY). 9.Kisko, J. L. & Barton, J. K. (2000) Inorg. Chem. 39, 4942-4949. 10.Jordan, P. & Carmo-Fonseca, M. (1998) Nucleic Acids Res. 26, 2831-2836.11. Fanou-Ayi, L. & Vijayalakshmi, M. (1983) Ann. N.Y. Acad. Sci. 413,300-306. 12. Hubert, P. & Porath, J. (1980) J. Chromatogr. 198, 247-255.13. Min, C. & Verdin, G. L. (1996) Nucleic Acids Res. 24, 3806-3810. 14.Hermann, T. & Westhof, E. (1998) Structure. 6, 1303-1314. 15. Gonzalez,R. L., Jr. & Tinoco, I., Jr. (1999) J. Mol. Biol. 289, 1267-1282. 16.Walter, F., Murchie, A. I., Thomson, J. B. & Lilley, D. M. (1998)Biochemistry 37, 14195-14203. 17. Sprat, B. G., Hedge, P. J., te, H. S.,Edelman, A. & Broome-Smith, J. K. (1986) Gene 41, 337-342. 18. Sioud, M.& Drlica, K. (1991) Proc. Natl. Acad. Sci. U.S.A 88, 7303-7307. 19.Murphy, J. C., Wibbenmeyer, J. A., Fox, G. E. & Willson, R. C. (1999)Nat. Biotechnol. 17, 822-823.20. Murphy, J. C., Fox, G. E. & Willson, R.C. (2001) Anal. Biochem. in press. 21. Cline, J., Braman, J. C. &Hogrefe, H. H. (1996) Nucleic Acids Res. 24, 3546-3551.22. Haupt, K.,Roy, F. & Vijayalakshmi, M. A. (1996) Anal. Biochem. 234, 149-154.

All references, patents, patent application or articles, cited hereinare incorporated by reference for all purposes permitted by controllinglaw and legal precedents. While this invention has been described fullyand completely, it should be understood that, within the scope of theappended claims, the invention may be practiced otherwise than asspecifically described. Although the invention has been disclosed withreference to its preferred embodiments, from reading this descriptionthose of skill in the art may appreciate changes and modification thatmay be made which do not depart from the scope and spirit of theinvention as described above and claimed hereafter.

8rtificialPCT Primer Sequence ttgc cgggaagcta gag 23225DNAArtificialPCTPrimer Sequence 2tcgcattgaa ttatgtgctg tgtag 2532ificialSyntheticOligonucleotide Sequence 3cagacgatag tcctagttgc 2ArtificialSyntheticOligonucleotide Sequence 4gtctgctatc aggatcaacg 2ArtificialSyntheticOligonucleotide Sequence 5aaaaaaaaaa aaaaaaaaaa 2ArtificialSyntheticOligonucleotide Sequence 6tttttttttt tttttttttt 2ArtificialSyntheticOligonucleotide Sequence 7cccccccccc cccccccccc 2ArtificialSyntheticOligonucleotide Sequence 8gggggggggg gggggggggg 2BR>

1. A method for separating compounds comprising the steps of: contactinga mixture comprising cell lysate or enzyme and a target polynucleotidecompound which includes at least four shielded purine or pyrimidinemoieties, and at least one contaminant polynucleotide compound whichincludes at least four non-shielded purine or pyrimidine moieties, andother compounds, with a solid composition including immobilized metalions capable of binding compounds containing a non-shielded purine orpyrimidine moiety, to form a liquid product containing a reduced amountof the contaminant polynucleotide and also at least 10% of the originalamount of the target polynucleotide compound which includes at leastfour shielded purine or pyrimidine moieties, substantially free fromhistidine tags.
 2. The method of claim 1 in which the solid compositioncomprises a polymeric material including a plurality of ligands bondedthereto, each ligand immobilizing a metal ion selected from the groupconsisting of Cu.sup.2+, Zn.sup.2+, Ni.sup.2+, Fe.sup.3+, Fe.sup.2+,Co.sup.2+, Sc.sup.2+, Ti.sup.2+, V.sup.2+, Cr.sup.2+, Mn.sup.2+,Ca.sup.2+, Cd.sup.2+ or Hg.sup.2+ or mixtures or combinations thereof.3. A method for separating compounds comprising the steps of: contactinga mixture comprising cell lysate or enzyme and a target protein, and acontaminant compound which includes at least four non-shielded purine orpyrimidine moieties, and other compounds, with a solid compositionincluding immobilized metal ions capable of binding compounds containinga non-shielded purine or pyrimidine moiety, to form a liquid productcontaining a reduced amount of the contaminant polynucleotide; andcollecting the target protein substantially free of polynucleotides. 4.A method according to claim 3 comprising collecting the target proteinwith a reduced content of polynucleotides.
 5. A method according toclaim 3 comprising collecting the target protein substantially free ofgenomic DNA.
 6. A method according to claim 3 comprising collecting thetarget protein substantially free of RNA.
 7. The method of claim 3 inwhich the solid composition comprises a polymeric material including aplurality of ligands bonded thereto, each ligand immobilizing a metalion selected from the group consisting of Cu.sup.2+, Zn.sup.2+,Ni.sup.2+, Fe.sup.3+, Fe.sup.2+, Co.sup.2+, Sc.sup.2+, Ti.sup.2+,V.sup.2+, Cr.sup.2+, Mn.sup.2+, Ca.sup.2+, Cd.sup.2+ or Hg.sup.2+ ormixtures or combinations thereof.
 8. A substrate comprising a pluralityof ligands bonded thereto, each ligand immobilizing a metal atom and/orion capable of binding compounds containing at least 4 non-shieldedpurine or pyrimidine moieties or groups, and a compound containing atleast 4 non-shielded purine or pyrimidine moieties or groups bound to aportion of the metal atoms and/or ions wherein the compound is selectedfrom the group of RNA, single stranded DNA, and other molecules havingat least 4 non-shielded purine and/or pyrimidine moieties or groups; orwherein the substrate comprises a polymeric material including aplurality of ligands bonded thereto, each ligand immobilizing a metalion selected from the group consisting of Cu.sup.2+, Zn.sup.2+,Ni.sup.2+, Fe.sup.3+, Fe.sup.2+, Co.sup.2+, Sc.sup.2+, Ti.sup.2+,V.sup.2+, Cr.sup.2+, Mn.sup.2+, Ca.sup.2+, Cd.sup.2+ or Hg.sup.2+ ormixtures or combinations thereof; or wherein the substrate is selectedfrom the group consisting of a polymer, a column inner wall, a membrane,an inorganic support, a metallic support, and a surface of an electronicchip.
 9. The method of claim 1 further comprising the step of:separating the liquid from the solid composition.
 10. A method accordingto claim 1 further comprising the steps of: separating the supernatantliquid from the solid composition; or further comprising the steps of:separating the supernatant liquid from the solid composition and elutingthe RNA and/or DNA including a non-shielded purine or pyrimidine moietyfrom the solid composition.
 11. Method according to claim 1 wherein themixture comprises poly(A) tailed mRNA sequences and other mRNA sequencesfrom eukaryotic cells, or wherein the mixture of compounds comprisesdenatured nucleic acid sequences, wherein sequences having A-richregions bind better than sequences having T-rich regions, so thatcomplementary strands can be resolved.
 12. A method of claim 1 whereinthe solid phase comprises a well plate, particle, magnetic particle,tube, pipette tip, monolith, or chromatographic packing.
 13. The methodof claim 1 wherein the contaminant compound comprises RNA having atleast four non-shielded purine and/or pyrimidine moieties and isseparated from a lysate containing double-stranded DNA.
 14. The methodof claim 1 wherein the contaminant compound comprises DNA having atleast four non-shielded purine and/or pyrimidine moieties and isseparated from a mixture containing double-stranded DNA and an enzyme.15. The method of claim 1 wherein the target polynucleotide compoundwhich includes at least four shielded purine or pyrimidine moieties ispresent in the original mixture at a concentration of less than 1micromolar.
 16. A method of claim 1 wherein the target compoundcomprises DNA.
 17. A method of claim 110 wherein the contaminantcompound comprises RNA.
 18. The method of claim 3 wherein the targetprotein is present in the original mixture at a concentration of lessthan 1 micromolar.
 19. The method of claim 3 further comprising the stepof: separating the liquid from the solid composition.
 20. A methodaccording to claim 3 further comprising the steps of: separating thesupernatant liquid from the solid composition; or further comprising thesteps of: separating the supernatant liquid from the solid compositionand eluting the RNA and/or DNA including a non-shielded purine orpyrimidine moiety from the solid composition.